CPY Document

L ry1 ~ :l~ I ! LIFE CYCLE EVOLUTION AND SYSTEMATICS OF CAMP ANULARIID ~ HYDROZOANS cß~~ By :;0+ Anette Frese Govindarajan

B.S., University of Connecticut, 1992 ...-- , M.S., University of Connecticut, 1994 MARINE BIOLOGICAL Submitted in partial fulfillment of the requirements for the degree of LABORATORY

Doctor of Philosophy LIBRARY + WOO HOLE, MASS. at the W. H. O. i. MASSACHUSETTS INSTITUTE OF TECHNOLOGY

and the

WOODS HOLE OCEANOGRAPHIC INSTITUTION 'fl ~ September 2004 \§ (Ç 2004 Anette Frese Govindarajan All rights reserved.

The author hereby grants to MIT and WHOI permission to reproduce paper and

electronic copies of this thesis in whole or in part and to distribute them publicly.

Signature of A Joint Program in Oceanography/Applied Ocean cIence and Engineering

Massachusetts Institute of Technology and Woods Hole Oceanographic Institution mber 2004 Certified by Laurence P. Madin Thesis Supervisor

Accepted by John Waterbury C a' , Joint Committee for Biology r Woo Hole Oceanographic Institution -:'l 'J

Acknowledgements

Many people generously contributed to all aspects of this thesis. First, I would like to thank my advisor, Larr Madin, for his support and encouragement throughout my time in the Joint Program. From the beginning, Ken Halanych graciously offered me use of his laboratory and welcomed me into his team, as well as technical and analytical advice. Cliff Cunningham generously provided funding and lab support as well as helpful advice on analyses. Jesús Pineda and Martin Polz also provided helpful advice at committee meetings. Nando Boero welcomed me into his lab in Italy and provided valuable insights into hydrozoan biology.

My thesis research involved working in several laboratories, and I am extremely grateful for all of the assistance I received. In Larr Madin's lab at WHOI, Erich Horgan and Nick Albanese were always helpful and provided good company. In Ken Halanych's lab at WHOI, Nan Trowbridge, Rob Jennings, and Yale Passameneck were also always helpful, especially with lab techniques, and provided good company. In Cliff Cunningham's lab at Duke University, my fellow PEET students Alberto Lindner and Maria Pia Miglietta were invaluable in discussions about hydroids. Also Bernie Ball, Christy Henzler, and Cynthia Reginos helped me around the lab and made me feel at home. In Nando Boero's lab at the Università di Lecce in Italy, Cinzia Gravili, Stefano

Piraino, Juergen Schmich, and Shin Kubota taught me a lot about hydro ids and made my visits there extremely enjoyable. Tim Shank at WHOI generously permitted me to finish my lab work in his lab after Ken Halanych moved to Auburn University. My research required samples from around the world, and many people generously contributed samples or helped me to collect. Nando Boero, Cinzia Gravili, and Stefano Piraino, as well as other members ofthe Boero laboratory, helped me collect samples from the Mediterranean. Nan Trowbridge helped me collect samples in California and Woods Hole, and Maria Pia Miglietta helped me collect samples in New Zealand. Rudi Scheltema and Ken Halanych brought me along on a cruise to Antarctica and helped me collect samples there. Ken Halanych helped me collect samples from

3 Friday Harbor. A. Collns, J. Coyer, C. Gravil, B. Grossman, A. Hart, L. Henry, Y. Hirano, E. Horgan, A. Lindner, i. Kosevich, S. Kubota, M. P. Miglietta, S. Piraino, K. Reise, P. Schuchert and N. Trowbridge also generously provided samples. Funding for my thesis was provided by WHOI Academic Programs, an NSF

PEET grant to Cliff Cuningham (DEB- 9978131), WHOI Ocean Ventures Fund, the Society for Integrative and Comparative Biology, WHOI Biology, and the MIT-Italy club. Finally, I would like to thank my family and friends for all their generous support throughout the past 6 years. My parents, sisters and their families, and the Trowbridges (my Falmouth family) provided support and sustenance. My husband Arvind provided encouragement and was always there for me. Thank you all!

4 Abstract

The purpose of this thesis is to study campanulariid life cycle evolution and systematics. The Campanulariidae is a hydrozoan family with many life cycle variations, and provide an excellent model system to study life cycle evolution. Additionally, the unique campanulariid Obelia medusae may have been "re-invented" from ancestors without medusae. Chapter 1 reviews campanulariid life cycles and taxonomy. Chapter 2 presents a phylogeny based on 18S rDNA, calmodulin, L6S rDNA and cytochrome c oxidase I (COI). Ancestral life cycles are reconstructed using parsimony. Medusa loss is common, and Obelia may derive from ancestors with typical medusae. Taxonomic results are discussed in Chapter 3. Bilardia, a nominal campanulariid, appears phylogenetic ally distant, while Bonneviella spp. (Bonneviellidae), are nested within the Campanulariidae. Campanulariid genera are not monophyletic. Orthopyxis integra and elytia gracilis may represent cryptic species, while Obelia longissima may be cosmopolitan. Chapter 4 investigates Obelia geniculata phylogeography. Japanese and North

Atlantic 16S rDNA and COI sequences are calibrated against the opening of the Bering Strait. Substitution rates are faster than in anthozoans and comparable to non-cnidarian invertebrates. Comparison of Pacific and Atlantic sequences suggests cryptic species exist. Finally, hydro ids in New Brunswick, Canada and Iceland may have survived the last glaciation.

5 6 TABLE OF CONTENTS

SIGNATURE PAGE Page 1

ACKNOWLEDGEMENTS Page 3

ABSTRACT Page 5

TABLE OF CONTENTS Page 7

CHAPTER 1. Introduction Page 9

CHAPTER 2. Independent origins of medusa loss in campanulariid hydrozoans Page 31

CHAPTER 3. New taxonomic insights on the Campanulariidae (Cnidaria, Hydrozoa)

Page 91

CHAPTER 4. Mitochondrial evolution and phylogeography in Obelia geniculata (Cnidaria, Hydrozoa) Page 117

CHAPTER 5. Conclusions Page 149

APPENDIX A. Species identification in Eugymnanthea Page 155

7 8 Chapter 1 Introduction

The Hydrozoa Cnidarians are known for their diversity of life cycles, and their evolution has been a subject of continuing debate for over a century (for example, Brooks 1886; Hyman 1940; Hadzi 1963; Schuchert 1993). Cnidarians are embodied by two basic forms: the polyp and the medusa. The Anthozoa have only the polyp stage, while the Scyphozoa, Cubozoa, and Hydrozoa (collectively termed the Tesserazoa; Salvini-Plawen, 1978 or the Medusozoa; Petersen, 1979) can have both the polyp and medusa stages.

Based on a combination of molecular and morphological data, Bridge et ai. (1992; 1995) showed that anthozoans are the basal cnidarians. The most parsimonious evolutionary scenario is that the ancestral cnidarian had a polyp-only life cycle, and that there was a single origin of the medusa in the ancestral medusozoan. The Hydrozoa is a group of cnidarians that may have both the polyp and medusa or only one or the other. Life cycle variation is the hallmark of the Hydrozoa (Boero et aI., 1997; Boero et aI., 2002). In the typically-presented hydrozoan life cycle, the sexual adult medusa releases either sperm or eggs, which fertilize and form short-lived, lecithotrophic (non-feeding) planulae (Figure lA). The planulae settle on to a substrate

and form larval hydroids (Boero and Bouilon, 1987). The hydro ids grow asexually, usually forming colonies. Through a complex process involving the entocodon, or medusary nodule (Boero et aI., 1998; Boero et aI., 2002), hydro ids produce and release medusae, completing the cycle. In many hydrozoans, however, gametes are released from medusoids (with some medusa features but not others, and mayor may not be released from the hydroid) or directly from the hydroid ("fixed gonophores" with no medusa features; Figure lB). The Campanulariidae (Hydrozoa, Leptomedusa) exhibit all ofthese variations on life cycle, and thus provide an excellent model system for the study of life cycle evolution.

9 The Campanulariidae The Campanulariidae (Leptomedusae) are an important, abundant, and widely distributed family ofhydrozoans (Cornelius, 1982). Members ofthe Campanulariidae are

common in planktonic and benthic environments around the world. The hydro ids grow on a variety of substrates, including rocks, seaweed, bivalve shells, and pilings and floating docks at marinas, and most can be easily found in intertidal and shallow subtidal areas. Campanulariids can be ecologically important; for example, in Georges Bank, pelagic colonies of the hydroid elytia gracils are important competitors for cod larvae

(Madin et aI., 1996). Campanulariids have also been used as model organisms to study bioluminescence (e.g., Morin, 1974, Markova et aI., 2002) and other aspects of physiology such as growth, aging, and stress responses (e.g., Toth, 1969; Brock, 1970;

Brock, 1974; Stebbing, 1981; Stebbing and Santiago-Fandino, 1983, Stebbing, 1985; Crowell, 1991; but see Hughes, 1987). Campanulariids are particularly interesting because members exhibit a variety of life cycles (Cornelius, 1982; Boero and Sarà, 1987; Boero et aI., 1996), providing a model system to study life cycle evolution (Table 1). For example, elytia and Obelia produce free medusae, Orthopyxis and Silcularia produce medusoids, Gonothyraea produce meconidia (unique medusoids never released from the hydroid), and eampanularia and Laomedea form fixed gonophores. The campanulariid Obelia, frequently used as a model hydrozoan in introductory biology classes, is morphologically and developmentally unique among the entire Hydrozoa (Kühn, 1913; Chapman, 1968; Boero et aI., 1996). Their unique featues include: 1) the presence ofhydroid-like chordal (solid), rather than medusa-like hollow, tentacles; 2) a "peduncled" manubrium (flared mouth) that resembles the hydroid hypostome (mouth); 3) the lack of a velum (inner ring of tissue around the bell margin) and true bell cavity resulting in a flattened shape; 4) statocysts positioned at the tentacle bases, rather than at the umbrellar margin; 5) lack of true tentacular bulbs; and 6) gonads that develop from the comers of the manubrium where the radial canals originate, rather

10 than directly from the radial canals (although they later migrate down the radial canals); and 7) cross-layered, rather than circular, myofibrils which are better suited to a flat, rather than the typical concave and craspedote form (Chapman, 1968). Obelia medusa development also differs from all other hydrozoan medusae, in that the entocodon or medusary nodule (a proliferation of ectoderm along the blastostyle that leads to the subumbrellar cavity) seems to disappear early in development (Kühn, 1913, Boero et aI., 1996).

Campanulariid gonophore development Gonophore development is relatively well-studied in the Campanulariidae (Berril, 1950; 1961). "Gonophore" refers to the strctures involved in sexual reproduction in species without medusae (Cornelius, 1995), but here the term is used to refer to the sexually reproducing body, regardless of whether it is released from, or fixed to, the hydroid. Typical medusa (e.g., elytia) development involves the formation of an entocodon, or medusary nodule (Figure 2A). The entocodon is formed along a modified polyp, termed the blastostyle. In elytia, medusae develop along only one side of the blastostyle. There is a thickening of the blastostyle epidermis, forming a nodule (the entocodon) between the apical peripheral epidermis and the adjoining endodermis. The entocodon invaginates to form a cavity which becomes the subumbrellar cavity in the medusa. The adjoining endodermallayer around the entocodon forms the radial canals, which eventually fuse at the distal end to form the circular canal of the medusa. The apical epidermis also gives rise to the tentacle chambers (note medusa tentacles are hollow), and a blastostyle protrusion of the endoderm and adjoining ectoderm into the nascent subumbrellar cavity eventually becomes the manubrium. As the blastostyle grows, the developing medusae are carred to the apical end of the gonotheca, and are liberated. Some unusual developmental variations are found in Gastroblasta (a nominal campanulariid genus now recognized as an aberrant elytia), which is similar to elytia except that the medusae have multiple manubria (mouths) and radial canals (Kramp, 1961; Boero, 1980). Another unusual variation is found in elytia mccradyi, where

11 blastostyle and medusa development takes place on the radial canals of medusae, where the gonads are usually located, rather than in hydroid gonophores; indeed, the existence of a hydroid stage in this species has yet to be confirmed (Carré et aI., 1995). This process is modified in species with medusoids and fixed gonophores (Berrll, 1950; Boero and Bouilon, 1989). In Orthopyxis, which produces medusoids, the blastostyle buds off medusoids with a reduced or absent manubrium, which are eventually liberated with the already matue gametes on well-defined radial canals. The medusoids may possess a velum (Hirohito, 1969). Only one or two medusoids are produced from the blastostyle, and the degree of development can differ between the sexes. The medusoid bud has a large entocodon and gonadal mass (Berrill, 1950). Medusoids that are not liberated from the hydroid may be further reduced, remain in gonotheca, and gametes are on the rudimentary radial canals. In Orthopyxis everta medusoids are not liberated and females expel their eggs into an acrocyst or external capsule located on top of the gonotheca (Nutting, 1915). Silicularia also produces sexually dimorphic medusoids in which the eggs develop along the radial canals and the planulae are brooded in females (Ralph, 1956; Blanco, 1967). The medusoids lack a manubrium and tentacles. There are no records of Silicularia medusoids being released, although that possibility has not been ruled out (Vervoort and Watson, 2003). The process of medusa formation is substantially different in Obelia (Figure 2B) (Kühn, 1913; Boero et aI., 1996). An entocodon is formed early in development, but subsequently appears to be lost (Figure 2B). Therefore, no velum or true subumbrellar cavity is formed (Figure 3). Development resembles hydranth development (Boero et aI., 1996). Thus, the Obelia medusa appears to be a chimera of the hydranth and medusa features (Boero et aI., 1996). Additionally, medusa development differs from elytia in that medusae can develop along all sides of the blastostyle, and the medusae are smaller and more numerous (Berrll, 1950). Gonothyraea shares some of Obelia's unusual features but not others. Weismann (1883) and Küh (1913) describe entocodon formation, but it apparently does not disappear as in Obelia (Berrill, 1950). The entocodon is larger than in Obelia and

12 differentiates mainly into gonad (Berril, 1961). Küh's (1913) drawings indicate that the meconidia tentacles are solid, endodermal polyp-like tentacles as in Obelia. Female meconidia possess more, longer, and more contractile tentacles than male me conidia, which assist in internal fertilization by trapping sperm (Miller, 1973). Female meconidia also have radial canals, which are absent in the males. All meconidia lack a functional manubrium (mouth) (Miler, 1973) and Boero et ai. (1996) consider it a spadix, the corresponding structue in fixed gonophores. In females, the hydrocoel currents into the abortive manubrium/spadix are maintained as the planulae develop, perhaps extending the meconidia lifespan (Berrill, 1950). Gonothyraea shares some similarities to elytia in that medusae bud off from only one side of the blastostyle, and developing medusae are in between elytia and Obelia in terms of number and size of medusa buds (Berrll, 1950). In fixed gonophore-bearing forms, such as Laomedea, the gonophores are further reduced and lack most medusa featues. Gametes surround the spadix. In Laomedea, gonophore development proceeds through a relatively large entocodon, which differentiates mainly into gonad, as in Gonothyraea (Berril, 1961). The sporosacs rupture and release gametes or planulae while in the gonangium. As in Obelia but unlike Gonothyraea and elytia, sporosacs bud from all sides of the blastostyle, and the number of buds is similar to Obelia although they may be larger. Details of development may vary between species. In Laomedea flexuosa, eggs remain in the female sporosac through development, fertilization, and planula development. In contrast L. calceolifera, eggs are "ovulated" from the sporosac but remain in the gonangium (Miler 1973). Interestingly, the monotypic Hartlaubella also "ovulates", but the eggs are pushed out of the gonangium and fertilization is external (Miler 1973). However, Vervoort and Watson (2003) report gonothecae containing developing planulae in New Zealand H. gelatinosa, so this may either be under environmental control or indicative of cryptic speciation. Another gonophore variation is found in L. inornata (Gonothyraea inornata) and L. neglecta, which produce external sacs, or acrocysts, that contain developing planulae (Nutting, 1915; Cornelius 1982; Chapter 3). Note the acrocysts in L. inornata are referred to as meconidia in the literature (as Gonothyraea inornata; Nutting 1901; Fraser 1946),

13 but because they have no medusoid featues they are more appropriately viewed as acrocysts (Nutting 1915; Chapter 3). Also only one acrocyst is extruded from the gonangium, in contrast to several meconidia in Gonothyraea. Gonophore development can be, to some extent, influenced by the environment. In some Orthopyxis integra, whether or not medusoids are released is under environmental control; however, the factors involved are unclear although season and hydrodynamic conditions are implicated (Cornelius 1982). Another campanulariid example is elytia linearis. In the summer, hydroids produce medusae which grow and reach sexual maturity through series of stages, but in the fall, medusae have matue gonads at birth, live only a few days, and do not attain adult somatic characteristics (Boero and Sarà, 1987). Cornelius (1990) considered reports that Hartlaubella gelatinosa could sometimes produce Obelia-type medusae instead of fixed gonophores, but the reports were inconclusive because of the possibility of misidentification due to difficulty distinguishing this hydroid from Obelia bidentata. Also as noted above, H. gelatinosa has been reported both to have internal (Vervoort and Watson, 2003) and external (Miler, 1973) fertilization; however, whether this is due to environmental factors or because they are actually different species remains to be seen. Finally, temperature could potentially affect medusa expression by affecting sex determination. Temperature has been shown to affect sex determination in elytia (Carré and Carré,

2000). If this holds true generally, then temperatue may consequently affect gonophore development, as many hydroids have sexually dimorphic gonophores, with the female exhibiting more medusoid features than the male.

Taxonomy Until recently, rigorous hypothesis testing of life cycle evolution in the Campanulariidae and other hydrozoans has not been possible because of difficulty in classification and species identification. Hydrozoan taxonomy is challenging at both the species level and higher because of diffculties resulting from their life cycles, distinguishing homology, and phenotyic plasticity. Unless an organism has been

14 cultued thoughout its life cycle, it has not been possible to link the hydroid and medusa stages (although now DNA technology has the potential to do this). Difficulties arose because specialists frequently examined only the hydroid or medusa stage. Consequently, there are many examples where the hydroid and medusa of the same species were given different names (Cornelius, 1977; Cornelius, 1982). For example, among the Campanulariidae, some elytia medusae were originally placed in a different genus, Phialidium, than their hydroids, and Orthopyxis medusae were originally placed in a different genus, Agastra, than their hydroids. Yet, even today, many species, including some campanulariids, remain described only from either the hydroid or medusa stage (Boero and Bouillon, 1993; Bouilon and Boero, 2000). Gonophore (life cycle) type is used, in part, to define genera in the Campanulariidae and other hydrozoan families (e.g., Cornelius, 1982). However, iflife cycle transitions occur often, then this approach could lead to paraphyletic genera (Boero et aI., 1996). Recent studies in anthomedusan hydrozoans suggest this may be the case (Petersen, 1990 in the Tubulariidae; Cunningham and Buss, 1993 in the Hydractiniidae). Another major diffculty challenging hydrozoan taxonomists is determining whether morphological differences are due to environmental or genetic causes

, (Cornelius, 1990). Early taxonomists described many new species based on small morphological differences. For example, Cornelius (1975) states that 70 species of Obelia were described between 1830 and 1948. More recently, authors have attributed many of these differences to environmental causes and combined many of the species. Accordingly, Cornelius (1990) only recognizes 4 species of Obelia in the eastern North Atlantic. However, other authors (Stepanjants, 1998; Kubota, 1999; Bouilon and Boero, 2000) recognize other species as valid in other parts of the world. In another example, there were 11 described species of Silcularia, distinguished primarily by variations in hydrothecallength. Ralph (1956), however, noted individual Silcularia colonies with hydrothecae spanning the range of sizes described for different species, and concluded that these differences were environmental and merged them into 3 species. As a result of

15 these and other redefinitions, many hydro ids now appear to have near-cosmopolitan distributions. Overall colony form and characteristics of the hydrotheca, or chitinous exoskeleton, are frequently used as taxonomic characters in thecate hydro ids such as the Campanulariidae (Cornelius, 1982). However, these features may exhibit considerable phenotypic plasticity (Ralph, 1956), and this presents a major obstacle to their taxonomic utility. Despite this, these characters form the primary basis for species descriptions in the

Campanulariidae. Alternatively, morphological characteristics of the nematocysts, or stinging capsules, can be taxonomically useful in many groups of hydroids, including campanulariids (Östman, 1979; Östman 1982; Östman, 1983; Gravier-Bonnet, 1987; Östman, 1987). But again, some nematocyst characters may be plastic (Östman et aI., 1987) and they are time consuming and require live material to study. Molecular genetic data have the potential to clarify both campanulariid taxonomy and evolutionary relationships, because markers relatively independent of life cycle and environment could be used. For example, Östman (1982; 1983) found inter- and intraspecific gel band differences for the enzme acid phosphatase in Scandinavian campanulariids, and suggested that taken with other characters, isoenzyme banding patterns could be taxonomically usefuL. There have been no additional enzyme studies, and no studies using DNA sequences, to date on campanulariids. Collection of molecular genetic data, such as DNA sequences, and subsequent phylogenetic analysis has become standard for many taxa, and could be applied to campanulariid hydroids. DNA sequences could be used to both distinguish species by identifying patterns of reciprocal monophyly

(A vise, 2000) and investigate higher level evolutionary relationships.

Phylogeography and mitochondrial evolution Phylogeography examines the role of historical processes in shaping modern species distributions in a phylogenetic framework (Avise, 2000). Mitochondrial DNA is often used in phylogeographic studies of invertebrates, because it is evolving at a faster rate than nuclear DNA and often provides resolution at the intraspecific level (Avise,

16 2000). However, mitochondral DNA may evolve exceptionally slowly in cnidarians, although previous studies are based primarly on anthozoans (Romano and Palumbi, 1997; Medina et aI., 1999; Van Oppen et aI., 1999; Shearer et aI., 2002). The only study on a hydrozoan used a relative rate test on COI amino acid sequences, and found that the hydrozoan (Limnomedusa) Maeotias sp. was evolving significantly more slowly than echinoderms, molluscs, and arthropods (Shearer et aI., 2000). The North Atlantic is an excellent system for phylogeographic studies because the organisms and recent geological history are relatively well known. Much of the Atlantic fauna and flora is believed to have originated in the Pacific, approximately 3.5 million years ago (mya), with the opening of the Bering Strait (Durham and MacNeil, 1967; Van den Hoek and Breeman, 1990; Vermeij, 1991, Cunningham and Collins, 1998). This migration is referred to as the trans-Arctic interchange, and is useful because it provides an upper bound for divergence estimates (Wares and Cunningham, 2001), especially for taxa like the campanulariids, which are not well represented in the fossil record. Mitochondrial DNA sequences from North Atlantic and North Pacific sister taxa can be analyzed so that substitution rates could be calibrated, the mitochondrial markers can be evaluated for their phylogenetic utility, and the recent history of the species can be elucidated.

Thesis goals The purpose of this thesis is to study life cycle evolution and systematics of the Campanulariidae. Chapter 2 presents a molecular phylogeny of the Campanulariidae, and tests hypotheses on life cycle evolution, including on the frequency of life cycle transitions and on the origin of the unique Obelia medusae. Chapter 3 discusses the taxonomic results of the molecular phylogeny presented in Chapter 2, and revises taxonomic diagnoses accordingly. Chapter 4 examines the mitochondral evolution and phylogeography of the widely distributed campanulariid, Obelia geniculata. The first calibrated hydrozoan substitution rates are presented. Finally, the appendix examines

17 species identification from molecular, morphological, and reproductive perspectives in the bivalve-inhabiting hydroid, Eugymnanthea (Hydrozoa, Leptomedusa, Eirenidae).

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systematics, ecology and evolution of hydro ids and hydromedusae. Clarendon Press, Oxford. pp. 67-82. Östman C, Piraino S, Roca I. 1987. Nematocyst comparisons between some Mediterranean and Scandinavian campanulariids (Cnidaria, Hydrozoa). In: Bouilon J, Boero F, Cicogna F, Cornelius, PFS. (eds). Modern trends in the systematics, ecology and evolution of hydroids and hydromedusae. Clarendon Press, Oxford. pp. 299-310.

23 Petersen KW. 1979. Development of coloniality in Hydrozoa. In: G. Larwood and B.R. Rosen (eds). Biology and systematics of colonial organisms. Academic Press, London. Petersen KW. 1990. Evolution and taxonomy in capitate hydroids and medusae (Cnidaria: Hydrozoa). Zool J Linn Soc 100: 101-231. Ralph PM. 1956. Variation in Obelia geniculata (Linnaeus, 1758) and Silcularia bilabiata (Coughtrey, 1875) (Hydroida, F. Campanulariidae). Trans R Soc NZ 84: 279-296. Romano SL, Palumbi SR. 1997. Molecular evolution of a portion of the mitochondrial L6S ribosomal gene region in sc1eractinian corals. J Mol Evol45:397-4l 1 Salvini- Plawen LV 1978. On the origin and evolution of the lower Metazoa. Zeit f Zool Syst Evol-Forschung 16: 40-88.

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24 Van den Hoek C, Breeman AM. 1990. Seaweed biogeography of the Nort Atlantic: where are we now? In: Garbary DJ, South GR (eds) Evolutionary biogeography

of marine algae in the North Atlantic. NATO ASI Ser G22, Springer Verlag, New

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25 Table 1. Campanulariid subfamilies and genera and their associated sexual stage. (gonophore type). Following Cornelius (1982), the three subfamily lineages are indicated. Bilardia, recognized by some (e.g., Vervoort and Watson, 2003), but not others (e.g., Cornelius, 1982) as a campanulariid, is here listed under the Campanulariinae. Following Calder (1991) and Boero et aL. (1996), Tulpa is placed in the Campanulariinae rather than the Clytiinae, as in Cornelius (1982).

Subfamily Genus Gonophore type Description Campanulariinae Orthopyxis Medusoids With some medusa features (i.e., radial canals) but not others (i.e., tentacles), some with extracapsular development (acrocysts); mayor may not be released from the hydroid

Silcularia Medusoids With some medusa features (i.e., radial canals) but not others; not known to be released from the hydroid

Campanularia Fixed gonophores Gonophores with no medusa featues; fixed on the hydroid

Rhizocaulus Fixed gonophores

Tulpa Fixed gonophores

Bilardia Fixed gonophores

Clytiinae Clytia Medusae Typical hydrozoan medusae

Obeliinae Obelia Medusae Atypical hydrozoan medusae

Gonothyraea Meconidia With some medusa features (i.e., tentacles), but not others; medusa features similar to Obelia, rather than typical, medusae; not released from the hydroid

Hartlaubella Fixed gonophores Gonophores with no medusa features; fixed on the hydroid

Laomedea Fixed gonophores Gonophores with no medusa features; some with extracapsular development (acrocysts); fixed on the hydroid

26 '"

o

Figure 1. Hydrozoan life cycles, adapted from Naumov (1960). A. Life cycle with a free medusa stage. The hydro ids produce and release medusae, which release gametes that fertilize and form planulae. The planulae settle and form hydroids. B. Life cycle where medusoids or fixed gonophores are produced instead of medusae. Medusoids mayor may not be released from the hydroid. Fertilization may be internal, and planulae may be brooded within the medusoids or fixed gonophores.

27 A.

B.

Figure 2. Hydrozoan medusae developmental progression in the tyical hydromedusa Syncoryne sarsii (A) and an Obelia medusa (B). The entocodon is indicated by arrows. Note the small entocodon and nascent subumbrellar cavity in Obelia disappear as development progresses. Images are from Kühn, 1913 and are oriented so that the tentacles are developing towards the top.

28 A.

Hollow t~ntrid~¡;

B. No bell cavity or vp.liim Solid t~ntrid~¡;

Figure 3. Cross section of a typical hydrozoan medusa (A) and an Obelia medusa (B). Note the flat shape, solid tentacles, and lack of a true bell cavity and velum in the Obelia medusa. Images are from Kühn, 1913 and are oriented so that the tentacles are pointed down.

29 30 Chapter 2 Independent origins of medusa loss in campanulariid hydrozoans*

* manuscript based on this chapter in preparation with Ferdinando Boero and Ken Halanych

Abstract The goal of this research is to understand the evolution of life cycles in campanulariid hydrozoans (Cnidaria, Leptomedusa). Life cycle diversity is a hallmark of the Hydrozoa, and the Campanulariidae provide an excellent model system in which to study hydrozoan life cycle evolution. Campanulariids, well known by their typically benthic hydroid stage, exhibit an extraordinary array of life cycles, ranging from species with a free medusa stage, to those with a reduced or absent medusa stage. Additionally, based on their unique morphology, the medusae of the campanulariid genus Obelia are hypothesized to have been derived from species lacking medusae, thus possibly representing an example of a rare evolutionary reversaL. However, inadequate taxonomic resolution has, up to now, prevented testing ofthis hypothesis. To complement previous morphological studies (which are hampered in a large part by their complex life cycles), a molecular approach was used to resolve campanulariid relationships, using 2 nuclear (18S rDNA and calmodulin) and 2 mitochondrial (16S rDNA and cytochrome c oxidase I

(COlD genes to construct a phylogeny of the Campanulariidae. The results indicate that life cycle transitions have occurred multiple times, and that Obelia medusae may be derived from ancestors with typical medusae, rather than medusoids or fixed gonophores, although this conclusion rests on the underlying phylogeny.

Introduction Marine invertebrates exhibit a diverse array of life cycles, often with multiple morphologically distinct stages separated spatially and temporally. The evolution of such complex life cycles is a subject of considerable study (e.g., Strathmann, 1978; Wray, 1996; McHugh, 1998; Hart, 2000). Many marine invertebrates with complex life cycles,

31 including echinoderms, bivalves, polychaetes and crustaceans, possess a sessile (or with limited mobility) benthic, sexually-reproducing adult stage that releases gametes. The gametes form free-living planktonic larvae, which after some period of time settle and metamorphose into adults. Frequently the larval stage may be shortened or absent

(Giangrande et aI., 1994).

Life cycle variation is a hallmark of the Hydrozoa (Cnidaria), and in particular, the Campanulariidae (Leptomedusa) exhibit considerable diversity in their reproductive strategies (Cornelius 1982; Boero and Sarà 1987; Boero et al. 1996), providing a model system to study life cycle evolution. Like other hydrozoans, many campanulariids (e.g., elytia, Obelia) have an asexual benthic polyp, or hydroid, stage that gives rise to a sexual adult planktonic medusa stage. The medusae in turn produce gametes that form short- lived lecithotrophic planulae, which settle and develop into hydroids. In contrast, other campanulariids (e.g., Laomedea, eampanularia) have apparently reduced or lost the medusa stage. Campanulariids display several stages of this putative evolutionary transition (Table 1). The range of complex life cycles, the frequency of medusa loss (e.g., Petersen 1990) and the possibility of "re-invented" medusae (e.g., Boero and Sarà, 1987) have fueled controversy on hydrozoan life cycle evolution and systematics. For example, there are many cases where the hydroid stage is placed in one taxon (species/genus/family etc.) and the medusa stage is placed in another (Boero, 1980). The decoupled rates of morphological evolution occurring in hydro ids and medusae (termed "inconsistent evolution" by Naumov (1960) and "mosaic evolution" by Rees (l957) and Morton (1957)) have been a major impediment to hydrozoan classification, and has prevented rigorous testing of phylogenetic hypotheses. Until relatively recently, separate classifications existed for hydro ids and medusae. Naumov (1960) was the first to present a unified system. Furhermore, some researchers (Allman 1864; Rees 1957; Millard 1975, Cornelius 1982, Vervoort and Watson, 2003) have either explicitly or implicitly assumed that loss or reduction of the medusa stage is a rare event and have used life cycle type to define genera. Others (Broch 1916; Kramp 1949; Petersen 1979; Petersen 1990;

32 Cunningham and Buss 1993; Boero et aI., 1996) have speculated that medusa loss happens relatively frequently, so that the use of life cycles as a generic character may have resulted in paraphyletic groupings. Finally, it is assumed that the presence of a medusa stage in hydrozoans is ancestral (Bridge et aI., 1995) and that medusae have been lost many times; Cornelius (l992a) estimates at least 60 episodes of medusa reduction.

However, the many unique features of the campanulariid Obelia medusae indicate that this may not always be tre, and suggest the possibility that some medusae may have been "re-invented" from forms lacking medusae (Boero and Sarà 1987; Boero et aI., 1996).

Obelia medusae Despite their prominence in textbooks as a model hydrozoan, Obelia medusae have morphological and developmental features that are unique among the Hydrozoa, (Kühn, 1913, Chapman, 1968; Boero et aI., 1996). These features include: 1) the presence ofhydroid-like chordal (solid), rather than medusa-like hollow, tentacles; 2) a "peduncled" manubrium (flared mouth) that resembles the hydroid hypostome (mouth); 3) the lack of a velum (inner ring of tissue around the bell margin) and true bell cavity resulting in a flattened shape; 4) statocysts positioned at the tentacle bases, rather than at the umbrellar margin; 5) lack of true tentacular bulbs; and 6) gonads that develop from the corners of the manubrium where the radial canals originate, rather than directly from the radial canals (although they later migrate down the radial canals); and 7) cross- layered, rather than circular, myofibrils which are better suited to a flat, rather than the typical concave and craspedote form (Chapman, 1968). Obelia medusa development also differs from all other hydrozoan medusae, in that the entocodon (a proliferation of ectoderm along the blastostyle that leads to the subumbrellar cavity) seems to disappear early in development (Kühn, 1913; Boero et aI., 1996). Interestingly, another campanulariid, Gonothyraea, produces unique medusoids, termed meconidia, whose medusa featues are like Obelia rather than typical medusae

(e.g., solid tentacles) (Boero et aI., 1996). Despite the striking features of their medusae

33 or medusoids, Obelia and Gonothyraea are placed within the Campanulariidae because of the strong similarity of their hydroids to other campanulariid hydroids. Thus, in addition to contributing to the understanding of medusa loss, the Campanulariidae offer a unique system to investigate the possibility of multiple acquisitions of the medusa form. Understanding this issue would fuher our knowledge of how bentho-pelagic life cycles

evolve relating to the early evolution of major animal lineages by providing insight into the rate of the evolution of development and the likelihood of evolutionary reversals.

Hypotheses of eampanulariid evolution Three leading hypotheses of campanulariid phylogeny and medusa evolution are shown in Figure 1 Cornelius (1982) divided the Campanulariidae into 3 subfamilies, and hypothesized a progression of medusa loss from medusae to medusoids/meconidia to fixed gonophores (Figue lA) In this scenario, Obelia is the ancestral obeliniid. Östman (1987; Figure 1B) suggested Gonothyraea was the ancestral obeliniid, based on the presence of a type of nematocyst also found in other Campanulariids but not Obelia. This hypothesis was based on Cornelius (1982) and Boero and Sarà (1987), the latter of whom were the first to suggest that Obelia medusae were secondarily derived. Boero et al. (1996; Figure LC) also modified the hypothesis ofBoero and Sarà to include additional morphological features and merged the Clytiinae into the Obeliinae, where it is basaL. The ancestral obeliniid in their hypothesis is Laomedea.

Goals

Although there has been considerable discussion of hydrozoan morphological and life cycle evolution, evolutionary hypotheses have never been tested in a rigorous manner against an independently derived phylogeny. The goal of this research is to understand life cycle evolution in the Campanulariidae. Towards this end, we have constrcted a molecular phylogeny based on 2 nuclear (18S rDNA and calmodulin) and 2 mitochondrial (16S rDNA and cytochrome c oxidase I (COlD genes to address the following questions: 1) In the Campanulariinae and Obeliinae lineages, did medusoids

34 and fixed gonophores evolve only once or many times? 2) Does Obelia derive from ancestors with typical medusae, medusoids, or fixed gonophores? Finally, we reviewed the ecological and evolutionary processes that have been implicated in generating hydrozoan life cycle diversity. In order to gain a broader view of campanulariid life cycle evolution, we examined our results in the context of marine invertebrate life history evolution.

Materials and Methods Sample collection and outgroup selection The taxa employed in this study and their collection localities are presented in Table 2. A total of 47 putative campanulariid taxa were obtained, including representatives of all recognized genera (except Hartlaubella and Tulpa). Due to the potential existence of cryptic species in some widely distributed forms, multiple representatives from different locations were included when available. Several thecate hydroids (Leptomedusae) were also collected to be used as outgroups (Table 2). In particular, the Bonneviellidae is thought to be closely related to the Campanulariidae (Bouillon, 1985). Additionally, several L8S rDNA sequences from GenBank were used primarily as outgroups (Table 3). Data collection

Field-collected hydro ids were identified and cut into two portions for preservation: one in 10% formalin for a morphological voucher, and one in 95% ethanol for DNA sequencing. Once in the lab, genomic DNA was extracted using DNEasy extraction kits (Qiagen) following the manufacturer's protocol. The entire 18S rDNA and portions of calmodulin, 16S rDNA, and COI were amplified using procedures and primers described in Halanych et al. (1998) for 18S rDNA, Lindner et al. (in prep) for calmodulin, Cunningham and Buss (1993) for 16S rDNA, and Folmer et al. (1994) for COI. PCR products were visualized on a 1 % agarose gel stained with ethidium bromide. In most cases, the entire gene (18S rDNA) or gene fragments (calmodulin, L6S rDNA, COI) were amplified in one reaction; however, in a few taxa, the L8S rDNA gene was

35 amplified in 3 shorter, overlapping segments. In some cases for calmodulin, the PCR product was re-amplified using 0.5 III template. The entire 18S rDNA bands were cut out of the gel and purified using the Qiagen PCR purification kit for gel purification, as occasionally multiple bands were present on the gel. In some cases, the purified PCR product was sequenced directly, while in others, it was cloned using the Promega T -GEM easy kit, and white colonies were screened for the insert by PCR amplification of a portion of the 1 8S rDNA and visualization on a 1 % agarose gel stained with ethidium bromide. Clones containing the insert were grown and purified using the Pro mega minipreps following manufacturer's protocol. The shorter 18S segments and the calmodulin, L6S rDNA, and COI were purified directly from the PCR product using the Qiagen PCR purification kit according to manufactuer's protocol. Purified PCR product was cycle-sequenced using either Big Dye 2 or 3 sequencing chemistry (ABI), following the manufacterer's recommendations. For the cloned L8S rDNA, the m13fand m13r primers, as well as 6 internal L8S rDNA primers (Halanych et aI., 1998), were used in sequencing reactions. In most cases, unincorporated dideoxynucleotides were removed with a Sephadex G-25 (Sigma, St. Louis, MO) column. The DNA was then sequenced on an ABI 377 or 3700 automated DNA sequencer. DNA sequences were aligned and checked for ambiguities using

Autoassembler (ABI) and Sequencher 4.2.2 (Gene codes Corp.). DNA sequences were aligned using ClustalX (Thompson et aI., 1994) and the alignments were confirmed by eye using MacClade (Maddison and Maddison, 2000). Regions that could not be unambiguously aligned from the rDNA were excluded from analyses. No sites were excluded from the calmodulin and COI sequences.

Phywgenetic anarys~ Because of uncertainties in hydrozoan systematics, a leptomedusan phylogeny was generated with the L8S rDNA sequences to confir rooting and delineate the

Campanulariidae. The Leptomedusae were rooted with two species of Hydridae (Collins, 2000; 2002) and the closest outgroups to the Campanulariidae were identified. Then, a

36 new alignment with all 4 markers and including only the Campanulariidae and the most appropriate outgroups was generated for subsequent analyses. The individual markers were analyzed both separately and together. The best-fit models for maximum likelihood analysis were determined with ModelTest (Posada and Crandall, 1998), and used in PAUP* 4.0blO (Swofford, 2002). Heuristic searches under maximum parsimony and maximum likelihood were conducted using starting trees obtained by stepwise addition with 10 random addition sequence replicates and TBR branch swapping. Support for nodes was obtained from parsimony and likelihood nonparametric bootstrap analyses and Bayesian posterior probabilities. 1000 bootstrap replicates were run when the parsimony criterion was used, while 100 bootstrap replicates were run when the likelihood criterion was used. In the parsimony bootstrap analyses, the maximum number oJ trees saved in each bootstrap replicate was set to 1000 (100 per addition sequencereplicate). To minimize computation time, maximum likelihood bootstrap analysis was only ru on the combined campanulariid datasets using one addition sequence replicate and limiting the number of rearrangements to 5000. Bayesian posterior probabilities were calculated with MRBA YES 3.0b4 (Ronquist and Huelsenbeck, 2003).

Bayesian analyses were run for 1,000,000 generations with 4 chains and a sample frequency of 1000. Burn-in was set at 200, and after analyses were ru, a plot of the log likelihoods versus generation was examined to confirm that the likelihood values had leveled off. Topological hypotheses were tested with the Shimodaira-Hasegawa (SH) test (Shimodaira and Hasagawa, 1999) implemented in PAUP* 4.0b10 (Swofford, 2002) using the RELL test distribution. Life cycle (gonophore) type was traced on to the parsimony bootstrap consensus tree of the combined dataset, and the ancestral states were reconstrcted using equally- weighted parsimony in MacClade 4.0 (Maddison and Maddison, 2000). Life cycle type was designated as tyical medusae, medusoids, meconidia, fixed gonophores, or Obelia medusae. It was assumed that the ancestral campanulariid possessed a typical medusa

37 stage. Because losses (e.g., going from medusae to medusoids or fixed gonophores) may be easier than gains (e.g., going from fixed gonophores and medusoids to medusae) and frequent losses may bias the results (Cuningham et aI., 1998; Cunningham, 1999), ancestral states were also reconstructed by creating a life cycle transition step matrix where gains were weighted greater than losses. Transition between medusa and medusoid types (typical/Obelia and medusoid/meconidia) were considered gains. A sensitivity analysis was conducted to see how sensitive ancestral state reconstruction was by comparing a range of weights applied to gains (e.g., Omland, 1997; Ree and Donoghue, 1998). Gain costs (weights) were tested at 0.1 increments between 1 and 2, and then periodically up to 99. Because Gonothyraea meconidia possess some of the unusual features found in Obelia medusae (e.g., solid tentacles and position of the gonads; Boero et aI., 1996) while Orthopyxis and Silcularia medusoids appear more like typical medusae (e.g., they may have a velum; Hirohito, 1969), ancestral state reconstruction was also conducted using only 3 life cycle categories: typical medusae/medusoids, Obelia medusae/me conidia, and fixed gonopohores.

Results L8S rDNA and L6S rDNA sequences were obtained from all specimens, and calmodulin and COI were obtained from most (Table 2).

Outgroup rooting and delineation of the eampanulariidae The leptomedusan phylogeny was based on an alignment including 2057 basepairs (bp) of 18S rDNA. 462 bp were excluded. Of the included sites, 434 bp were variable and 255 bp were parsimony-informative. The results clearly show that the Campanulariidae are not monophyletic (Figure 2). Bilardia subrufa falls well outside the primary "campanulariid" clade. Bilardia, Stegella, and Melicertissa form a clade and, along with the sertlariids Gymnangium and Selginopsis, fall basal to the Campanulariidae. There is a highly supported clade

38 composed of the campanulariids (except Bilardia), Aequorea, Blacliordia, Eugymnanthea, Tiaropsidium, Opercularella, and ealycella. Lovenella gracils and Eucheilota bakeri fall inside the campanulariid clade. The bonneviellids were strongly supported to be deeply nested in the Campanulariidae, in the Campanulariinae. The SH test results were consistent with the phylogenies: inclusion of Bilardia in the Campanulariidae and exclusion ofthe bonneviellids were significantly less likely, while the inclusion of Eucheilota and Lovenella in the Campanulariidae was not significantly less likely (Table 4).

Relationships in the eampanulariidae Based on Figure 2, Eugymnanthea, ealycella, and Opercularella were selected as outgroups for the expanded Campanulariid phylogeny. Lovenella, Eucheilota, and the bonnevielliçls were also included because a close relationship with the Campanulariidae was indicated, while Bilardia was excluded because it appeared phylogenetically distant. A total of3108 bp could be aligned in the combined dataset (18S: 1612 bp; CAM: 401 bp; 16S: 434 bp; COI: 661 bp). 262 bp of 18S and 176 bp of L6S were excluded. 890 bp were variable (18S: 359; CAM: 125; L6S: 131; COI: 275), and 673 were parsimony- informative.(18S: 216; CAM: l07; L6S: 105; COI: 245). In the two protein-coding genes, most of the variation occurred in 3rd codon positions (110/125 variable sites in CAM and 208/275 in COI). The data were analyzed both together (Figure 3) and separately (Figures

4-7), and indicated that most of the phylogenetic signal came from the 18S rDNA sequences, while the others were generally very poorly resolved (by parsimony). Therefore the combined and L8S rDNA results are emphasized below. The combined and L8S analyses yielded similar results, with a few exceptions noted. Maximum likelihood, parsimony, and Bayesian support often agreed, although in some cases the Bayesian posterior probabilities were considerably higher than the likelihood and parsimony bootstrap values. Because Bayesian posterior probabilities may sometimes overestimate support (Erixon et aI., 2003; Simmons et aI., 2004), only nodes with that also had high bootstrap values were considered well-supported.

39 The Campanulariidae, inclusive of Bonneviella spp. and exclusive of Eucheilota bakeri and Lovenella gracilis, appears monophyletic but this clade is not well supported (Figure 3, Figure 4). Eucheilota and Lovenella fall immediately basal to this clade, and these taxa plus the campanulariids and bonneviellids form a very highly supported clade. Within the Campanulariidae, there are 4 major lineages (the Campanulariinae, elytia, and 2 Obeliinae lineages) that are well-supported, with the exceptions of elytia hummelincki and Obelia bidentata, which are hard to place. The arrangement of these major lineages with respect to each other is not well resolved. Monophyly of the Campanulariinae, including the bonneviellds, was very strongly supported (Figure 3, Figure 4). Monophyly ofthe campanulariniid genera were not well-supported, however. Orthopyxis appears to form a monophyletic clade in Figure 3 (but not Figure 4), but this clade does not have either bootstrap or Bayesian support. Bonneviella also appears to form a monophyletic clade in Figure 3 (but not Figue 4), which is highly supported by Bayesian, but not bootstrap, analyses. eampanularia is not monophyletic in either Figure 3 or 4. In the combined analysis, Orthopyxis everta and Orthopyxis integra Italy (IT) group together strongly (Figure 3), while in the L8S rDNA analysis, Orthopyxis everta and eampanularia hincksii group together strongly. The grouping in the combined analysis is probably due to the strong support of that arrangement in the 16S rDNA (Figure 6) and COI (Figue 7). elytia appears monophyletic in the combined dataset, although the inclusion of elytia hummelincki, in the basal position, has only moderate support (Figure 3). Without elytia hummelincki, support is higher. In the L8S rDNA, elytia hummelincki falls outside the main elytia clade, although its position there is not supported (Figure 4). The Obeliinae, although, may be paraphyletic. In the combined data, Obelia bidentata plus the elytia clade form a sister group to the rest of the Obeliinae (Figure 3). This position is only minimally supported, however. Both parsimony and Bayesian analyses weakly support the placement of Obelia bidentata in the Obeliinae, as the basal member of the clade containing Laomedea flexuosa, Laomedea inornata, and Obelia longissima (values of72 and 60, respectively; this arrangement not depicted in Figure 3).

40 In the Bayesian analysis, when the 4 genes and codon positions for the protein-coding genes are partitioned, the original arrangement in Figure 3 is supported only by a posterior probability of 57. Parsimony analysis of the 1 8S rDNA data alone highly supports this position (bootstrap value of95; Figure 4). With the possible exception of Obelia bidentata, the Obeliinae form two well- supported clades (Figue 3; Figure 4). One consists of Gonothyraea loveni, Obelia dichotoma, Laomedea calceolifera, and Obelia geniculata, and the other consists of Laomedea flexuosa, Laomedea inornata, and Obelia longissima. Thus, the obeliniid genera are not monophyletic. The SH tests were again consistent with the phylogeny: trees constrained to have monophyletic Obelia and Laomedea were significantly less likely, while there was no significant difference between the most likely tree and trees constrained to have monophyletic Obeliinae and eampanularia (Table 4).

Life cycle evolution and origin of Obelia Obeliniid and possibly campanulariniid genera were not monophyletic (Figure 3; Figue 4, Table 4), indicating that life cycles evolved multiple times. Because both parsimony bootstrap and Bayesian analysis supported (albeit weakly) a basal position of Obelia bidentata (which had exceptionally long branches) in the obeliniid Laomedea flexuosa, Laomedea inornata, and Obelia geniculata clade, in contrast to the likelihood topology (tree) which placed it as the basal elytia (Figure 3), ancestral state reconstruction analysis was conducted on both the likelihood topology (Figure 3) and a modified topology, with Obelia bidentata placed in the obeliniid clade (e.g., Figue 13). The 18S rDNA also strongly supported the position of Obelia bidentata in the modified topology (Figure 4), and the SH test showed that a monophyletic Obeliinae was not significantly less likely (Table 4). A heuristic maximum likelihood analysis of the combined dataset with Obelia bidentata removed yields a topology otherwise identical to Figure 3.

41 In the likelihood topology, equally-weighted ancestral state reconstruction suggested that the ancestral campanulariid and obeliniid + elytia could not be determined (i.e., equivocal), and that the ancestral campanulariniid and obeliniid had fixed gonophores (Figue 8). A gain cost between 1.1 and 1.4 resulted in the ancestral campanulariid and obeliniid + elytia having typical medusae (Figure 9). Increasing the gain cost to 1.5 and then to 1.6 - 1.9 again resulted in the ancestral campanulariid and obeliniid + elytia being equivocal (Figure 10; Figure 11), although for gain costs 1.6-1.9, the ancestral obeliniid appeared to have an Obelia-type medusa (Figure 11). Gain costs of 2.0 and higher gave similar results, except that the ancestral campanulariniid appeared equivocal (Figure 12). In the modified topology, equally-weighted ancestral state reconstruction indicated that the life cycles of the ancestral campanulariid, elytia, and obeliniid were equivocal, and the ancestral campanulariniid had fixed gonophores (Figure 13). However, when the gain cost was increased to 1 1, the ancestral campanulariid and elytia had typical medusae, the ancestral obeliniid had Obelia medusae, and the ancestral campanulariniid had fixed gonophores (Figure 14). Increasing the gain cost had no further effect until it reached 2.0 (Figure 15). In this case, the ancestral campanulariid and elytia had tyical medusae, the ancestral obeliniid had Obelia medusae, and the ancestral campanulariniid was equivocaL. Increasing the gain cost up to 99 had no fuher effect. To see how the underlying phylogeny might also affect ancestral state reconstrction in the Campanulariinae, the modified topology was modified further, placing eampanularia hincksii in the clade with eampanularia volubils, Rhizocaulus, and the bonneviellids (Figue 16). In this case, even when there was no gain cost, the ancestral campanulariniid was equivocaL. Equally-weighted ancestral state reconstrction using 3 life cycle categories (tyical medusae/medusoids, Obelia medusae/meconidia, and fixed gonophores) on the modified topology (with Obelia bidentata placed within the Obeliinae) indicated that the ancestral campanulariid and obeliinid + elytia had typical medusae/medusoids, the ancestral obeliinid had Obelia medusae/meconidia, and the ancestral campanulariniid was

42 equivocal (Figure 17). Some ancestors within the Obeliiniae were equivocaL Gain costs of l. 1 - 99 yielded the same result except that the ancestral campanulariniid had typical medusae/medusoids, and no ancestors within the Obeliinae were equivocal (Figure 18).

Discussion The results corroborate some aspects of previous classification but refute others.

A detailed discussion of the taxonomic results is outside the scope of this chapter, but can be found in Govindarajan and Boero (chapter 3).

Multiple origins of medusa loss and the origin of Obelia Our results indicate multiple life cycle transitions in the Obeliinae and possibly the Campanulariinae. These results are similar to those in the Tubulariidae (Petersen, 1990) and Hydractiniidae (Cunningham and Buss 1993), suggesting that medusa reduction may be more common than once thought, and adding to the growing list of multiple losses of life history traits in marine invertebrates in general (Hart, 2000). Molecular studies in other hydrozoan lineages wil be helpful in revealing the extent of this phenomenon. In the maximum likelihood topology, when Obelia bidentata it is placed in the elytia, under some weighting schemes (gain cost = 1.0 - l.4) the ancestral obeliniid appears to have fixed gonophores, and Obelia medusae appear to have evolved multiple times. Alternatively, at higher gain costs, the relevant ancestors are equivocaL. Under these scenarios, either Obelia medusae would have to be evolved independently more than once, or elytia medusae would have had to been derived from Obelia medusae. Both possibilities indicate that medusa "re-invention" may be easier than previously thought. However, Obelia bidentata has extremely long branches in the maximum likelihood topology, which might have resulted in its erroneous placement. Maximum likelihood bootstrap does not support this position, and both parsimony and bootstrap analyses support, albeit weakly, its position as the basal member of one of the obeliniid

43 clades. Parsimony analysis of the L8S rDNA alone also strongly supports (95 percent) this position and the SH test indicated that this topology is not significantly less likely than the most likely topology. Thus, caution is warranted in interpreting the ancestral state reconstruction based on this topology as the majority of the support suggests Obelia bidentata is misplaced. Ancestral state reconstruction on the likelihood topology modified to reflect the alternative position of Obelia bidentata yielded very different results. Under equally- weighted parsimony, the ancestral forms of the Campanulariidae, Obeliinae, and elytia could not be inferred, but when even a very small gain cost was applied, the ancestral campanulariid and obeliniid + elytia appeared to have typical medusae. This result suggests that Obelia medusae derived directly from an ancestor with tyical medusae, rather than one with fixed gonophores or meconidia. This scenario is also consistent with a single origin of Obelia medusae, which makes sense in light of morphology, as all Obelia medusae are nearly identical (Kramp, 1961, Cornelius, 1995). It appears likely that Obelia medusae derived directly from typical elytia-like medusae and that the fixed gonophore bearing Laomedea evolved multiple times from Obelia. This result is also found when life cycle types are placed in 3, rather than 5, categories, and is inconsistent with all hypotheses of campanulariid evolution presented in Figure 1. One potential caveat, however, is that not all known obeliinids with fixed gonophores, especially Hartlaubella (a monotypic genus), could be obtained for this study. Like Laomedea, Hartlaubella has fixed gonophores; however, hydroid colony morphology differs in some potentially important ways. Hartlaubella hydro ids are relatively large, polysiphonic, and have toothed hydro thecal margins. These characteristics are generally similar to those found in Gonothyraea and some Obelia and elytia hydro ids but not Laomedea. Unless gonophores are present, Hartlaubella is easily confused with Obelia bidentata (Cornelius 1990). Additional studies are needed to determine if these featues indicate a basal position in the Obeliinae. This possibility does not affect the conclusions that the obeliniid genera are not monophyletic and that Obelia medusae were likely lost multiple times.

44 It is not possible to determine whether fixed gonophores evolved once or multiple times within the campanulariniid lineage. Analyses of both campanulariid topologies indicated that at no or low gain costs (1.0 - 1.9), the ancestral campanulariniid had fixed gonophores, but at higher gain costs (2:2.0), the ancestral form was equivocaL. Ifthe lower gain costs are correct, it would suggest that the medusoids of Silcularia and Orthopyxis are an example of an evolutionary reversaL. Again, this conclusion rests on the underlying topology: when eampanularia hincksii, whose placement is not supported by parsimony bootstrap, is moved into the eampanularia/Rhizocaulus/Bonneviella clade, the ancestral campanulariniid appears equivocal even at no gain cost. Additional caution should be exercised in interpreting the campanulariniid results because they may reflect bias in taxon sampling. All members of the very highly supported campanulariinid/onneviellid clade were collected in the North Pacific, consistent with a local, geographically restricted radiation there; in particular; all bonneviellids are found only in a few locations in the North Pacific (except Bonneviella grandis, which is also found in the North Atlantic (Naumov, 1960)). In contrast, eampanularia hincksii, which was the only other eampanularia in this study, was collected from the Mediterranean and is a nearly cosmopolitan species (Cornelius, 1995).

Other ean;panularia species exist in other parts of the world (e.g., Milard, 1975) that could notbe obtained for this study, but are necessary to determine the frequency of life cycle transitions in this lineage.

Evolutionary mechanisms of medusa loss The results suggest that if gains are even slightly more likely than losses, medusae may have been reduced multiple times in the Obeliinae (modified topology; Figure 14). Heterochrony, or change in the timing of development, is likely the underlying cause of medusa reduction. Weismann (1883; summarized in Berrll and Liu, 1948) related gonophore tye to the location of germ cell differentiation. In free-living medusae, the germ site is on the ectoderm in the medusa. As medusa features are lost, the germ site moves to the entocodon (medusa bud) and gonophore endoderm and eventually to the

45 hydroid coenosarc, or tissue. The germ cells then migrate to the gonophore ectoderm, where they matue, perhaps blocking medusa development (Boero and Bouilon, 1989). This phenomenon has been interpreted as paedomorphosis, and may occur via neoteny (retardation of somatic development) in large hydroids or progenesis (acceleration of sexual development) in small hydroids (Boero et al. 1997).

The results also indicate that Obelia medusae may not be derived from me conidia or fixed gonophore - bearing ancestors, and so are not an example of an evolutionary reversaL. Dollo' s law contends that complex strctues, such as many larval and medusoid features, are more easily lost than gained (Futuyma, 1998). There are few documented exceptions to Dollo's law; these include insect wings (Whiting et aI., 2003), mammalian molar teeth (Werdelin 1987), arthropod eyes (Oakley and Cunningham, 2003), and gastropod shell coiling (Collin and Cipriani, 2003). Evolutionary reversals in life history mode include planktotrophy in littorinid gastropods (Reid 1989). Recently Collin (2004) suggested that evolutionary reversals in developmental mode may have possibly occurred multiple times in calyptraeid gastropods, although her results rested on the assumptions of ancestral state reconstrction.

In a survey of marine invertebrate larval feeding structures, Strathmann (1978) found that planktotrophy, once lost, was unlikely to be regained, although it may be more likely in spiralian (molluscs, annelids, echiurans, sipunculans, entoprocts) than oligomeric (lophophorates, hemichordates, echinoderms) taxa. He also pointed out that reacquired planktotrophy would be less likely to have reduced egg size and thus greater fecundity (a benefit usually associated with planktotrophy). From a theoretical viewpoint, Ebenman (1992) argues that a life cycle stage, once lost, would be unlikely to be regained through gradual evolution, because there would be no juvenile-adult selection tradeoff in the derived direct developing phenotype. Finally, from a genetic perspective, when a complex strcture (like a life-cycle stage) is lost, the genes involved are not subject to selection and degrade over time unless they are used in other functions (Marshall et aI.,

1994). Thus re-expression can occur if there is rapid speciation and divergence times are short enough that the genes are not lost. Within a rapidly radiating lineage, a complex

46 trait can "flicker" on and off. Alternatively, conservation of gene integrity through functioning in different pathways could also allow the re-expression of a lost structure (Marshall et aI., 1994). This can happen for meristic traits in the same life cycle stage, or via heterochrony, when the traits are expressed in a different life cycle stage in the ancestor (Colln and Cipriani, 2003), as in hydrozoans. The re-evolution of medusae is considered unlikely because the medusa is a complex individual, comprising multiple physiological systems working together to perform all life functions necessary for existence - feeding, locomotion, reproduction, etc. In this regard it is not simply the re-emergence of an organ (the gonophore), but of an entity that must function as a whole self-sufficient individuaL. Our results suggested that Obelia, despite its unique features, was probably not a re-expressed medusa, although the possibility that it arose from an ancestor with meconidia or fixed gonophores cannot be completely ruled out. Futue studies wil need to determine the mechanism of Obelia's unique medusa features. Their similarity in some characteristics to hydranths may indicate that hydranth developmental pathways could be involved. Gene expression studies could be useful in evaluating this hypothesis. There may be, however, other cases of a secondarily derived planktonic stage in hydrozoans - termed "swimming gonophores", rather than medusae, by Boero and Bouilon (1989), because they lack most medusoid features that typicalleptomedusan medusae and medusoids and Obelia medusae share, while possessing some featues of fixed gonophores. The potential existence of re-expressed hydrozoan medusae or swimming gonophores could be due to any combination of the following mechanisms (adapted from Marshall et aI., 1994): 1) the re-expressed medusae diverged relatively recently from fixed gonophore ancestors, and the necessary genes have not been dormant long enough to degrade; 2) the genes involved may have other functions in the hydroid; and 3) changes in developmental timing, such as in gamete matuation, may allow medusa development to complete. Dating species divergences (e.g., Govindarajan et aI., submitted), gene expression, and detailed developmental studies wil help distinguish between among possibilities.

47 Ecological correlates and framework for future research Decades of research have examined the range of developmental modes for marine benthic invertebrates with planktonic larvae (Thorson, 1950; Mileikovsky 1971; Vance, 1973; Strathmann, 1985, Wray and Raff, 1991). Larvae range from being planktotrophic (feeding) and spending several weeks in the plankton, to lecithotrophic (nonfeeding) and spending a short time (minutes to days) in the plankton, to direct development, or brooding within the benthic adult. This continuum is analogous to the range of gonophore types seen in hydrozoans, except that in hydrozoans, it is the adult, not the larval, stage that is planktonic. As in the Campanulariidae, loss of the planktonic stage appears to be frequent in marine invertebrates with complex life cycles. For example numerous independent losses of planktonic feeding larvae have been hypothesized in echinoids (Emlet, 1990; Wray, 1996); asterinid starfish (Hart et aI., 1997); and gastropods (Lieberman et aI., 1993; Duda and Palumbi, 1999). The primary difference between hydrozoans and other marine invertebrates is that in hydrozoans it is (usually) the adult rather than the larval stage that is lost; however, the common loss of the planktonic stage suggests that hydrozoans and other marine invertebrates could be responding to common selective pressures. Dispersal ability is frequently considered in studies of life history evolution of marine invertebrates, although in many cases it appears not to be an important factor. Planktotrophy is generally associated with greater dispersal and larger geographic ranges

(e.g., Jablonski 1986; Strathmann, 1986; Scheltema 1989; Emlet 1995) álthough studies comparing genetic differentiation at different spatial scales give mixed results, and indicate the relationship is not clear-cut (Jackson, 1986; Palumbi, 1995) and hydrodynamic and behavioral factors could limit dispersal (e.g., Hamner et aI., 1994).

Greater dispersal may be a consequence, rather than a cause, of plank to trophy. Holoplankton, which ostensibly disperse in their adult stage, also can have planktotrophic larvae (Strathmann, 1985). And beyond a certain point, additional time in the plankton may not increase dispersaL. For example, Scheltema (1989) found that among gastropods,

48 planktotrophs had larger geographic ranges, but there was no difference in geographic range between those planktonic for only a few weeks and those planktonic for a few months or more. Among hydrozoans, species with medusae may also disperse farther than species without them (Boero, 1984). Medusae can live between a few weeks to a few months (Arai,1992), and so have the potential to travel far. On the other hand, hydrodynamic or behavioral processes could limit medusa movements (Arai, 1992; Hamner et aI., 1994). If the presence of a medusa stage were important for dispersal, then species with a free- living medusa stage may have greater distributions than those without a medusa. However, Boero and Bouilon (1993) showed that for Mediterranean hydrozoans whose life cycles are known, the percentage of species with a cosmopolitan distribution is significantly higher for species without a medusa than those with a medusa. Within the campanulariid Obeliinae, the lineage that includes members both with and without free medusae, it appears that species with medusae have larger geographic ranges than those without (Table 5). All Obelia species have near cosmopolitan distributions, while none of the Laomedea, Gonothyraea or Hartlaubella do. However, anthropogenic introductions and cryptic speciation may be confounding factors, so this conclusion should be regarded as tentative. It is possible that all Obelia "species" represent crytic species complexes. In a controversial taxonomic revision, Cornelius (1975) synonymized some 120 nominal taxa into 3, and later revised that number to 4 (1990). Others suspect his revisions went too far and recognize additional species (e.g., Kubota, 1999). In this study, Obelia longissima from widely separated locations appeared to have very little genetic divergence, indicating it may be trly cosmopolitan (Chapter 3). In contrast, Govindarajan et aI. (Chapter 4, submitted) showed that despite a high dispersal potential, there was significant genetic strcture and evidence for cryptic speciation in Obelia geniculata. Additional genetic studies wil be necessary to define species boundaries in other taxa. A confounding factor in studying the relationship between developmental mode and dispersal is that the planktonic larvaV medusa stage is not the only life cycle stage

49 that can disperse. Benthic adults (Highsmith 1985; Ingolfsson, 1995; Hobday, 2000) and hydrOlds (Kramp 1927; Cornelius 1992b; Calder, 1995; Ingolfsson 1995) can grow on seaweeds which could transport them over long distances and thus provide an alternative dispersal mode. Calder (1995) found medusa and fixed gonophore species to be equally likely to be represented on Sargassum, but others found that species with fixed gonophores were more likely on algal substrates. In hydrozoans, dispersal may also occur via planulae, which can live anywhere from about a few hours up to a few weeks (Bodo and Bouillon, 1968; Sommer, 1992). Although contrary to what might be expected if a planktonic period were important for dispersal, planulae in medusa-bearing species are longer-lived than in species with fixed gonophores (Sommer, 1992). Hydrozoans also may produce asexual propagules or frstules (Billard 1904; Berril 1948) that could disperse. Frustules are formed in some Obelia, and could provide an alternative dispersal mode when environmental conditions preclude medusa development (Panteleeva, 1999) or perhaps in species without medusae. Among benthic invertebrates, an extended planktonic period could be deleterious. Pelagic larvae are thought to be exposed to high mortality, due to predation, starvation, and dispersal away from suitable settling habitats, although it is difficult to quantify

(Morgan, 1995). In particular, too much dispersal can carr organisms outside ofthe range of tolerable environmental conditions and away from potential mates, especially for species whose hydro ids live in specialized coastal habitats (Edwards 1973) or remote islands (Cornelius 1992b; Calder, 1993). For example, in the "Paradox of Rockall" hypothesis, species with long planktonic periods are unlikely to persist in remote islands (Johannesson, 1988). While they may be able to initially colonize, their larvae may not be retained in sufficient concentration to maintain the population. In contrast, once a species with a reduced or lacking planktonic stage reaches a remote island, perhaps through algal rafting, it is more likely to persist because its larvae are not lost. Among hydrozoans there are several examples consistent with the paradox of Rockall, suggesting overdispersal is a real possibility. Cornelius (1992a) showed that in the relatively isolated oceanic islands ofthe Azores, there is a high proportion of

50 hydro ids with no medusa stage, and that these likely arrived to the islands by rafting. He suggested the medusae released from hydroids in remote islands would likely be lost to sea (Cornelius, 1992a). In this case, hydro ids with fixed gonophores may reproduce more successfully than those with a medusa stage. Calder (1993) and Kirkendale and Calder

(2003) similarly found a relatively higher proportion of fixed gonophore species in Bermuda and Guam, respectively. Similarly, Calder (2000) found the majority of species from 3 seamounts to have fixed gonophores. Developmental mode is related to adult size, with planktotrophy associated with larger adults and brooding associated with smaller adults (Strathmann and Strathmann, 1982). These authors suggest 3 potential hypotheses to explain the relationship: 1) allometry - brooding space may not be proportional to adult size, and larger adults are less capable of retaining and ventilating their offspring; 2) recruitment variability - larger, longer-lived adults can afford the riskier, but potentially more rewarding, strategy of plank to trophy; and 3) dispersal-larger adults receive greater larval dispersal benefits. Among thecate hydroids, a relationship between hydroid size and developmental mode is observed (of course, exceptions exist), with relatively larger hydroids associated with brooding, and relatively smaller hydroids associated with medusae (Cornelius, 1990). This trend appears opposite to that in other invertebrates. Cornelius (1990) argued that smaller and presumably shorter-lived or annual hydroids wil have only one chance to produce planulae, and it may be more advantageous for them to have a medusoid form so that planulae are spread over a larger area. Larger and presumably longer-lived and perennial hydroids wil have many chances to produce planulae that wil be able to successfully settle locally. Unlike most other thecates, in the Obeliinae, the relationship may be more like other marine invertebrates, with the larger hydro ids more likely to release planktonic medusae (Table 5). Obelia are generally larger than Laomedea, although Hartlaubella and Gonothyraea are comparable. More studies on hydroid longevity and recruitment are necessary. Developmental mode may also be linked to resource allocation (Vance, 1973; Strathmann, 1985). Planktotrophic species produce more, but smaller, eggs than those

51 with non-feeding development, resulting in higher fecundity. However, planktotrophy is also associated with higher mortality due to the longer pelagic period. Those with lecithotrophic or direct development have a larger parental investment and produce larger, but fewer, eggs that spend less or no time in the plankton. From models, Vance (1973a,b) concluded that the optimal developmental strategy depended on the relative importance of planktonic predation, abundance of planktonic food, and the length of time spent in the plankton, with planktotrophy favored when planktonic predation is low and planktonic food is high. A general correlation between marine invertebrate developmental mode, latitude, and water depth was first noticed by Thorson (1950), and termed "Thorson's rule" by Mileikovsky (1971). Thorson's rule suggests that pelagic development is prevalent in littoral and sublittoral zones in the tropics and subtropics, while it is considerably less common in high latitudes and deeper water. These trends may be related to the tradeoffs between energy allocation and the hazards of planktonic existence, with lower food supply and temperatures (increasing developmental time) in polar and deep sea regions (Vance, 1973). Many exceptions to Thorson's rule have been documented, however, and phylogenetic and ecological constraints may affect distributional patterns (e.g., Gallardo and Penchaszadeh, 2001). In hydrozoans, there is also some evidence for a correlation of medusoid production with latitude. Stechow (1924) found that among athecate hydrozoans, species with fixed gonophores were more prevalent in polar regions and deep seas, while those with free medusae were prevalent in warmer areas. The Eutimidae, like the

Campanulariidae, is also composed of members with medusae (Eutima) and medusoids (Eugymnanthea), and while they have overlapping distributions, Eugymnanthea is only found in the northernost of these localities (Kubota 1987). Kubota (1987) suggested that cooler temperatures during the Pleistocene may have driven the evolution of Eugymnanthea, because Eutima medusae cannot mature at low temperatues. Furthermore, in the Obeliinae, while the cosmopolitan Obelia is found in polar waters, it persists there primarily asexually, through frstule production in the hydroid, because the

52 water is too cold for medusa development (Panteleeva, 1999). Hartlaubella, Gonothyraea, and Laomedea are usually not found in tropical waters (with the exception of Laomedea pseudodichotoma). Additionally in the Campanulariidae, as discussed above, season (which is correlated with temperature) is related to medusa development in elytia linearis, with reduced planktonic medusa development in the fall compared to the summer (Boero and Sarà, 1987). A comparison of Campanulariid distributions indicates that species without medusae are more likely to be found in higher latitudes or deeper water. Within the Obeliinae, Laomedea, Gonothyraea, and Hartlaubella are found primarily in temperate and boreal regions, with Laomedea pseudodichotoma, found only off the tropical west coast of Africa (Vervoort, 1959), as the sole exception. In contrast, Obelia and elytia are represented in warmer waters. In the Campanulariinae, all Bonneviella species are found in the North Pacific (B. grandis is also in the North Atlantic), especially in deep (lOO's of meters) water (Nutting 1915; Naumov 1960). Rhizocaulus and Tulpa are also found exclusively in colder waters (Vervoort, 1972; Cornelius, 1995). eampanularia and Orthopyxis are widespread (Cornelius, 1995), while Silcularia is only found in cooler waters of the southern hemisphere. While it is clear that the causes and consequences of developmental mode evolution in campanulariids and other hydrozoans are still speculative, comparisons with other invertebrates could provide new insights. While hydrozoans differ in that it is their adult rather than larval stage that is modified, it is important that in both groups the planktonic stage has been reduced multiple times, possibly due to common selective pressures. The diversity of life cycles is likely driven by multiple factors, including allometry, temperature, and resource utilization. Dispersal appears to be less important, but future studies are necessary to sort out the relative contribution of these and potentially other factors. Evaluating ecological shifts in a phylogenetic framework, using the comparative method, wil be particularly insightfuL. eonclusions

53 The first molecular phylogeny of the Campanulariidae is presented. Genera are not monophyletic, indicating that life cycle transitions have occurred multiple times within the Campanulariidae. Specifically, within the Obeliinae, medusae may have been reduced to meconidia and fixed gonophores multiple times and the unique Obelia medusae may have been derived from an ancestor with typical medusae. These conclusions rest on the underlying phylogeny and the assumption that gains of strcture are less likely than losses. Additional research is necessary to corroborate these findings, and to understand the ecological correlates associated with the different life cycle types.

Acknowledgements We are grateful to numerous people, including A. Collins, J. Coyer, C. Gravili, B. Grossman, A. Hart, L. Henry, Y. Hirano, E. Horgan, A. Lindner, i. Kosevich, S. Kubota, M. P. Miglietta, S. Piraino, K. Reise, P. Schuchert and N. Trowbridge for collection assistance and/or providing samples. C. Cunningham and L. Madin provided valuable advice throughout the duration of this project. A. Govindarajan was supported by WHOI Academic Programs and an NSF PEET grant (DEB--9978131) to C. Cunningham. Additional research funds were provided by WHOI Ocean Ventures Fund, Society for Integrative and Comparative Biology, and the MIT-Italy club.

54 Table 1. Campanulariid subfamilies and genera and their associated sexual stage. (gonophore type). * = monotyic genus; ** including Gastroblasta.

Subfamily Genus Gono~ore ~ Description Campanulariinae Orthopyxis Medusoids With some medusa features (i.e., radial canals) but not others (i.e., tentacles), some with extracapsular development (acrocysts); mayor may not be released from the hydroid

Sileularia Medusoids With some medusa features (i.e., radial canals) but not others; not known to be released from the hydroid

Campanularia Fixed gonophores Gonophores with no medusa features; fixed on the hydroid

Rhizoeaulus* Fixed gonophores

Tulpa Fixed gonophores

Bilardia Fixed gonophores

Clytiinae Clytia** Medusae Typical hydrozoan medusae

Obeliinae Obelia Medusae Atypical hydrozoan medusae

Gonothyraea* Meconidia With some medusa features (i.e., tentacles), but not others; medusa features similar to Obelia, rather than typical, medusae; not released from the hydroid

Hartlaubella* Fixed gonophores Gonophores with no medusa features; fixed on the hydroid

Laomedea Fixed gonophores Gonophores with no medusa features; some with extracapsular development (acrocysts); fixed on the hydroid

55 Table 2. Campanulariid taxa and outgroups used in this study, collection locality, and genes sequenced.

Campanulariidae Taxon Locality 188 calmodulin 168 COI - rDNA rDNA Campanulariinae Bilardia subnifa Antarctic X X peninsula Campanularia Otranto, Italy X X X X hincksii Campanularia Monterey, CA, X X X X volubilis USA Orthopyxis everta Torre del X X X Serpe, Italy Orthopyxis Italy X X X X integra Orthopyxis Friday Harbor, X X X integra W A, USA Orthopyxis Monterey, CA, X X integra CA sp. I USA Orthopyxis Monterey, CA, X X X integra CA sp. 2 USA Orthopyxis Aleutians, USA X X X X integra Orthopyxis Sandgerdi, X X X integra Iceland Orthopyxis New Zealand X X X X integra Orthopyxis Brazil X X X X sargassicola Rhizocaulus Aleutians, USA X X X X verticllatus Silcularia rosea Bay ofIslands, X X X New Zealand Clytiinae Clytia Brazil X X X X elsaoswaldae Clytia gracils Georges Bank, X X X X North Atlantic Clytia gracilis Italy X X X X Clytia gracilis Beaufort, NC, X X X X USA Clytia gracils Woods Hole, X X X X MA, USA Clytia North Sea X X X X hemisphaerica Clytia S. Caterina, X X X X hummelincki Italy Clytia South Africa X X X X hummelincki Clytia linearis Brazil X X X Clytia linearis Torre X X X Inserraglio, Italy Clytia linearis Beaufort, NC, X X X X USA Clytia noliformis Brazil X X X

56 Clytia paulensis Otranto, Italy X X X X Clytia sp. CA, USA X X X X Obeliinae Gonothyraea Dennis, MA, X X X loveni USA Gonothyraea Roscoff, France X X X lovenii Laomedea Woods Hole, X X X X calceolifera MA, USA Laomedea Roscoff, France X X X X flexuosa Laomedea Iceland X X X X flexuosa Laomedea White Sea X X X X flexuosa Laomedea Friday Harbor, X X X X inornata W A, USA Obelia bidentata Beaufort, NC, X X X X USA Obelia bidentata North Sea X X X X Obelia dichotoma Otranto, Italy X X X X Obelia geniculata Roscoff, France X X X X Obelia geniculata New X X X X Brunswick, Canada Obelia geniculata Japan X X X X Obelia geniculata New Zealand X X X X Obelia longissima Ryders Cove, X X MA, USA Obelia longissima Antarctic X X X X peninsula Obelia longissima Dunedin, New X X X X Zealand Obelia longissima Sandgerdi, X X X X Iceland Obelia longissima White Sea X X X Outgroups Bonneviellidae Bonneviella regia Aleutians, USA X X X X Bonneviellidae Bonneviella sp. 2 Aleutians, USA X X X X Bonneviellidae Bonneviella sp. 3 Aleutians, USA X X X X Bonneviellidae Bonneviella sp. 4 Aleutians, USA X X X X Campanulinidae Calycella syringa Woods Hole, X X X X MA, USA Campanulinidae Stegella lobata Antarctic X X peninsula Eirenidae Eugymnanthea Taranto, Italy X X X X tnquilina Eucheilotidae Eucheilota bakeri CA, USA X X X Lovenelldae Lovenella gracilis Wildwood X X Crest, New Jersey Phiallelidae Opercularella Woods Hole, X X X pumila MA, USA

57 Table 3. Hydrozoan taxa with 1 8S rDNA from GenBank and associated accession numbers. ehlorohydra and Hydra belong to the Hydndae, and the remainder belong to the Leptomedusae.

Family Species Accession number Hydndae Chlorohydra viridissima AF358081 Hydridae Hydra circumcincta AF358080 Aequoreidae Aequorea aequorea AF358076 Aequoreidae Aequorea victoria AF358077 Aglaopheniidae Gymnangium hians Z86122 Blackfordiidae Blackfordia virginica AF358078 Campanulariidae Clytia sp. AF358074 Laodiceidae Melicertissa sp. AF358075 Sertulariidae Selaginopsis cornigera Z92899

Tiaropsidae Tiaropsidium kelseyi AF358079

58 Table 4. Results of the Shimodaira - Hasegawa tests for monophyly at the family and generic levels. The best tree (unconstrained) was compared with the best tree under the given constraints.

Constraint Monophyly tested -In L best -In L constraint Dirr -In L P value Family I Campanulariidae, as defined with Bilardia, the 7442.80525 7547.97789 105.17264 0.000 bonneviellds and Eucheilota and Lovenella Family 2 Campanulariidae, as defined without Bilardia, 7442.80525 7514.35024 71.54499 0.001 the bonneviellids, and Eucheilota and Lovenella Family 3 Campanulariidae, as defined without Bilardia, 7442.80525 7446.50419 3.69894 0.255 and Eucheilota and Lovenella, but with the bonneviellids Family 4 Campanulariidae, as defined with Eucheilota 7442.80525 7485.02453 42.21928 0.009 and Lovenella, and without Bilardia and the bonneviellds Obeliinae Obelia + Laomedea + Gonothyraea 20426.69624 20426.85739 0.16110 0.483 Obelia Obelia 20426.69624 20767.61421 340.91797 0.000 Laomedea Laomedea 20426.69624 20746.78747 320.09123 0.000 Campanularia Campanularia + Rhizocaulus + Bonneviella 20426.69624 20431.6449 4.66825 0.294 (Campanulariinae with fixed gonophores)

59 Table 5. Obeliinae ecological features summarized from Cornelius (1982; 1995), Millard (1975), Chaplygina (1993), Nutting (19l5), Blanco (1968), Fraser (1946), Vervoort (1959), Vervoort and Watson (2003). * may be the result of an anthropogenic introduction. Colony size represents maximum.

~~ies Range Hydroid colony features Obelia bidentata Nearly cosmopolitan Up to 350 mm, branching, polysiphonic Obelia dichotoma Nearly cosmopolitan Up to 350 mm, branching, mono- or polysiphonic Obelia geniculata Nearly cosmopolitan Up to 40 mm, branching, monosiphonic Obelia longissima Nearly cosmopolitan Up to 350+ mm, branching, usually monosiphonic Gonothyraea loveni North Atlantic, North Pacific, Up to 100 mm, branching, Australia, New Zealand, South monosiphonic Africa* Hartlaubella North Atlantic, Mediterranean, Up to 200 mm, branching, gelatinosa New Zealand, Patagonia, NE polysiphonic Pacific Laomedea angulata NE Atlantic, Mediterranean Up to 15 mm, unbranched, monosiphonic Laomedea calceolifera North Atlantic, Mediterranean, Up to 20 mm, unbranched, Brazil, South Africa*, North monosiphonic * Pacific Laomedea exigua NE Atlantic, Mediterranean, Puget Up to 4 mm, unbranched, Sound* monosiphonic Laomedea flexuosa North Atlantic, North Pacific* Up to 30 mm, sometimes branching, monosiphonic Laomedea inornata Puget Sound, China, Argentina Up to 36 mm, branching, monosiphonic Laomedea neglecta North Atlantic Up to 25 mm, branching, mono- or bisiphonic Laomedea Tropical West Africa Up to 50 mm, branching, pseudodichotoma part polysiphonic

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70 A. ci '" l -i %l ~ ~ 1 l. ~'" '" ã ~ lJ .. c3 cs Ô'

B.

c.

l¡ .g .~ "" ~ E: "" ií ~ .. 11 E; ~ ~ .~ C; BJ -'g c3 cs õ'

Figure 1. Proposed relationships among the Campanulariidae, reflecting 3 possibilities for the evolution of Obelia. A. Obelia derived from tyical medusae (adapted from Cornelius, 1982); B. Obelia derived from fixed gonophores, which derived from meconidia (adapted from Östman, 1987); C. Obelia derived from fixed gonophores (adapted from Boero et aI., 1996). Cornelius (1982) and Boero et ai. (1996) considered Silcularia to have fixed gonophores. However, they have medusoids (Ralph, 1956; Blanco, 1967) and their figues are modified here. All hypotheses show a single loss of medusae in the Campanulariinae and Obeliinae lineages. Finally, Boero et ai. considers elytia to be part ofthe Obeliinae rather than a separate lineage as in Cornelius (1982) and Östman (1987).

71 10011001I oCholorohydra viridissiiia Hydra cireuiicine/a Bilardia subrifa Siegel/a lobala Melieer/issa sp.

I OOil 00/ i 00 GylinCliigium liaiis Selagiiiopsis eornigera Calycella syringa 60/192 89'91'100 . ., , Opercularella pumila noil 00 Tiaropsidium kelsyi Eiigynman/hea iiiqidliiia Blackfordia virginica JOOIl OO/JOO Aequorea aeql10rea Aeqorea vie/oria Smeularia rosea

95/1001100 Ortliopyxis integra - AK

61/1 Campctniilaria98/94/1°ll hineksii Ort iopyxis everta 160/ Cctmpanularia volubi/is 58íí0 Boniicviel/a regia 0/85íl O( Rliizoeaiiliis vertcil/atus 100/97/100 66//63 Boiineviella sp. 2 99íl Lovenel/a gracilis Eucheilo/a bakeri Goiiotliyraea loveni - MA o 5f! 00 /67/10094 Obelia 4f! oÇJbelia.geliiculata dicllOtoma - FR 5 ¡ 3/86 Laomedea ealeeolifera Obelia bidentata - NC Laometlea j1exuo,~a - FR Obelia loiigissima - MA 93/100/100 Laomedea iiiornata Clyria liuJlmelilieki - IT

clyia paiilensis

Clyria sp.

Clytia 1Ì1learis clytia gracilis - GB Clytia liemisphaeriea - 0.01 substitutions/site Figure 2. Maximum likelihood phylogeny of the Campanulariidae plus additional Leptomedusae and Hydridae sequences, including those from Genbank. Putative campanulariids are indicated in bold. The phylogeny is from a heuristic search based on L8S rDNA sequences. The best-fit model selected by Modeltest was TrN + I + G. The -In likelihood for the topology = 7442.80525. First number refers to maximum likelihood bootstrap, second number refers to parsimony bootstrap, and third number refers to Bayesian posterior probability. Location codes: AN=Antarctic peninsula; AS=Aleutians; BR=Brazil; CA=California; FR=France; GB=Georges Bank; IC=Iceland; IT=Italy; JP=Japan; MA=Massachusetts; NB=New Bruswick; NJ=New Jersey; NZ=New Zealand; NC=Nort Carolina; NS=North Sea; SA=South Africa; W A=Washington.

72 Figure 3. Maximum likelihood phylogeny from a heuristic search based on the combined L8S rDNA, calmodulin, 16S rDNA, and COI sequences. The best-fit model selected by Modeltest was GTR + 1+ G. The -In likelihood for the topology = 20426.3370. First number refers to maximum likelihood bootstrap, second number refers to parsimony bootstrap, and third number refers to Bayesian posterior probability. Subfamily lineages are indicated. Location codes are as in Figure 2. Gonophore type indicated in bold after the taxon name. T = typical medusae; D = medusoids; F = fixed gonophores; M = meconidia, 0 = Obelia medusae.

73 11 00 Eugymnanthea inquílna D Calycella syringa F 98/78/100 Opercularella pumila F 100/99/1 00 Lovenella gracils T Eucheilota bakeri T Campanularia hincksii F Campanularia volubils F 100/100/100 Rhizocaulus verticilatus F Bonneviella sp. 4F Bonneviella sp. 2 F Bonneviella regia F Campanulariinae 52//96 Bonneviella sp. 3 F Silcularia rosea D (+ Bonnevielldae) 96/ 1/ 100/99/100 Orthopyxis everta D Orthopyxis integra IT D 53//93 OrthopyxÎS integra CA 1 D Orthopyxis integra NZ D //56 Orthopyxis sargassicola D Orthopyxis integra WAD Orthopyxis integra CA2 D Orthopyxis integra AS D Orthopyxis integra LC D 59//88 100/100/1 00 Obelia bidentata NC 0 I Ob l" Obelia bidentata NS 0 e nnae 100/100/1 00 Clytia hummelincld SA T Clytia hummelincki IT T Clytia paulensis T //80 Clytia gracilis IT T Clytia gracilis GB T 96/80/100 97/55/100 Clytia nolijòrmis T 100/100/100 Clytia linearis BR T Clytiinae 84/65/100 Clytia linearis IT T 100/1 00/1 00 Clytia linearis N C T 91/52/100 Clytia gracilis MA T Clytia hemisphaerica T /53/59 Clytia gracilis NC T Clytia elsaoswaldae T 99/100/100 Clytia sp. T Gonothyraea loveni MA M Gonothyraea loveni FR M Laomedea calceolifèra F Obelia dichotoma 0 100/99/100 I 00/1 00/1 Bbelia geniculata FR 0 57/99/95 //61 Obelia geniculata NB 0 99/94/100 Obelia geniculata JP 0 85/67/100 Obelia geniculata NZ 0 Obeliinae 100/100/100 Laomedea flexuosa FR F Laomedeaflexuosa IC F 100/1 00/1 00 100/100/100 Laomedeaflexuosa WS F Laomedea inornata F Obelia longissima AN 0 99/1 00/1 00 Obelia longissima WS 0 84/96/89 Obelia longissima IC 0 Obelia longissima NZ 0 Obelia longissima MA 0 - 0.01 substitutions/site

74 100/100 Eugymnanthea inquilna 9611 00 Calycella syringa Opercularella pumila 0/97 Loveiiella gracilis Eucheilota bakeri 89/98 Goiiothvraea loveni MA Goizothl'raeå loveni FR /69 " Obelia dichotoma Laomedea calceolirera Obeliinae Obelia geniculala NB Obelia geniculata FR /70 Obelia geiiiculata JP Obelia geniclilata NZ Orihopyxis integra IT Silcularia rosea 1001100 Bonneviella sp. 4 9lí 00 Rhizocaulus veriicillatiis Bonneviella sp. 3 Bonneviella sp. 2 Campanularia voliibils 57/96 Bonneviella regia Campanulariinae 96/ 00 Orthopyxis everla Campanularia hincksii (+ Bonneviellidae) Ortliopyxis sargassicola Ortliopyxis integra CA 1 Orthopyxis integra NZ /76 Orthopyxis integra AK Orthopyxis integra ie /88 Oriliopyxis integra CA2 /76 Orthopyxis integra W A Clytia gracilis IT 51. Clytia elsaoswaldae 94/100 Clytia sp. '9 Clytia gracilis WH Clytia hemispliaerica Clytia paulensis Clytia gracilis OB Clytiinae qvtia nolifonnis Clytia gracilis NC 58/99 Clytia linearis BR Clytia linearis IT Clylia linearis NC 100/100 Clvtia hummelincki SA Clytia hummelincki IT 100/100 Obelia bidentata NC Obelia bidentata NS 1001100 Laomedea.fexuosa FR 95/ /74 Laomedeaflexuosa IC Laomedeaflexuosa WS 100/100 Obelia longissima NZ Obeliinae Obelia longissima MA Obelia longissima WS 100/99 Obelia longissima IC Obelia longissima AN Laoniedea inornata - 0.005 substitutions/site

Figure 4. Maximum likelihood topology based on 18S rDNA sequences alone. The best- fit model selected by Modeltest was TIM + I + G. The -In likelihood of the topology = 6469.86106. Numbers before the slash refer to parsimony bootstrap values and numbers after the slash refer to Bayesian posterior probabilities. Subfamily lineages are indicated. Location codes are as in Figure 2.

75 Opercularella pumila Campanularia volubils Campanularia hincksii 54/97 /85 Rhizocaulus verticilatus Bonneviella sp. 2 Bonneviella sp. 4 55/9 Bonneviella regia /55 Bonneviella sp. 3 Campanulariinae Silcularia rosea /91 Orthopyxis sargassicola (+ Bonneviellidae) Orthopyxis integra W A Orthopyxis integra AK Orthopyxis integra CA sp. 1 Orthopyxis integra IT Ortliopyxis integra NZ Eucheilota bakeri Laomedea./exuosa FR lOOIl 00 Laomedea jlexuosa IC /78 Laomedea jlexuosa WS Laomedea inornata Obelia longissima AN 79/100 Obelia longissima NZ 100/82 Obelia longissima WS 62/72 67/95 Obelia longissima IC Gonothyraea loveni MA Obe1iinae Gonothyraea loveni FR Obelia dichotoma Laomedea calceolifera 54/99 93/9bbelia geniculata FR Obelia geniculata NB 7lí86 Obelia geniculata IP 67/100 Obelia geniculata NZ Clytia paulensis I Clytiinae 69/100 Obelia bidetnata NC I Obelia bidentata NS Obe1iinae Clytia gracilis MA Clytia hemisphaerica Clytia gracils IT Clytia gracilis NC Clytia elsaoswaldae Clytia sp. Clytia gracilis GB Clytiinae Clytia noliformis Clytia linearis BR 100/100 Clytia linearis IT /91 Clytia linearis NC 100/100 C(vtia humme/incld SA /76 Clytia humme/incki IT /91 Eugymnanthea inquilna Calycella syringa - 0.01 substitutions/site

Figure 5. Maximum likelihood topology based on calmodulin sequences alone. The best- fit model selected by Modeltest was TrN + I + G. The -In likelihood of the topology = 2975.97532. Numbers before the slash refer to parsimony bootstrap values and numbers after the slash refer to Bayesian posterior probabilities. Subfamily lineages are indicated. Location codes are as in Figure 2.

76 Opercularella pumila Eugymnanthea inqui/na Silcularia rosea Campanularia hincksii Bonneviella sp. 4 97/100 Rhizocaulus verticilatus Bonneviella sp. 3 Campanularia volubils Bonneviel/a regia Bonneviel/a sp. 2 Orthopyxis integra CA 1 Campanu lariinae 100/100 Orthopyxis everta Orthopyxis integra IT (+ Bonneviellidae) Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra CA2 Orthopyxis integra W A Orthopyxis integra AK 62/99 Ortliopyxis integra IC 77/100 Lovenel/a gracilis Euchei/ota bakeii Clytia gracilis IT Clytia gracils GB Clytia paulensis Clytia no!i/òrmis 100/100 Clytia hummelincki SA Clytia hiimmelinc/ä IT Clytia gracils MA Clytiinae qytia hemisphaerica /55 1O01l 00 Clytia ¡¡nearis BR 88/100 100/100 Clytia linearis IT Clytia linearis NC Clytia gracils NC qvtia e!saoswaldae Clytia sp. CaZvcella syringa Obelia dichotoma Obelia geniculata FR /99 Obelia geniculata NB Laomedea calceolirera Obelia geniculata JP Obelia geniculata NZ GonothyTaea loveni FR Gonothyraea loveni MA 901 i 00 Obelia bidentata NC Obelia bidentata NS Obeliinae

100/100 LaomedeaLaomedea.fexiiosa .fexiiosa IC FR Laomedea.fexuossa WS Laomedea inornata Obelia longissima MA Obelia longissima NZ Obelia longissinia WS Obelia longissima IC Obelia longissima AN - 0.05 substitutions/site

Figure 6. Maximum likelihood topology based on 16S rDNA sequences alone. The best-

fit model selected by Modeltest was GTR + 1+ G. The -In likelihood of the topology = 2981.05837. Numbers before the slash refer to parsimony bootstrap values and numbers after the slash refer to Bayesian posterior probabilities. Subfamily lineages are indicated. Location codes are as in Figure 2.

77 Eugymnantliea inquilna Calcycella syringa Clytia liiiearis NC Clytiinae Laomedea calceolirera 9líl00 Obelia dichotoma Obelia geniculata JP Obelia geniculata NZ Obelia geniculata FR Obelia geniculata NB Obeliinae 10011 00 Laomedea jlexuosa FR Laomedea flexuosa IC 73/100 100/100 Laomedea jlexuosa WS Obelia longissima NZ 100/100 Obelia longissÜna WS

90/100 Obelia longissima IC Obelia longissima AN Obelia bidentata NC Obelia bidentata NS Clytia gracils IT CZvlia paulensis 100/100 C(ytia Iiummelincki SA Clytia hummelincki IT Clytiinae Clytia elsaoswaldae Clytia sp. Clytia gracils NC Clylia gracilis MA Clylia hemisphaerica Orthopyxis sargassicola 196 1 o Oil 00 Orthopyxis everta Orthopyxis integra IT Ortliopyxis integra AK Orthopyxis integra IC Campanulariinae Ortliopyxis integra NZ Campanularia hincksii Clylia gracils GB I Clytiinae Rliizocaulus verlicillatus Campanularia volubils 58/100 Bonneviella sp. 4 Bonneviella sp. 2 Campanulariinae Boiiieviella regia (+ Bonnevielldae) Bonneviella sp. 3 - 0.05 substituions/site

Figure 7. Maximum likelihood topology based on COI sequences alone. The best-fit model selected by Modeltest was GTR + I + G. The -In likelihood ofthe topology = 6877.61370. Numbers before the slash refer to parsimony bootstrap values and numbers after the slash refer to Bayesian posterior probabilities. Subfamily lineages are indicated. Location codes are as in Figure 2.

78 Eugymnanthea inquilna Ca7ycella sy'ringa Opercularella pumila Typical medusae Lovenella gracilis Eucheilota bakeri Medusoids Campanularia hincksii Campanularia volubils Meconidia Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 .. Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 1,\tØ6?r.:1 Equivocal Silcularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CA i Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopy.xis integra IC Obelia bidentata NC Obelia bidentata NS Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracils GB Clytia noliformis Clytia linearis BR Clytia linearis IT Clytia linear is NC Clytia gracilis MA Clytia liemisphaerica Clytia gracilis NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomeåea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Laomedea flexuosa FR Laomedeal1exuosa IC Laomedeajlexuosa WS Laomedea inornata Obelia longissima AN Obelia longissima WS Obelia longissima NZ Obelia longissima MA

Figure 8. Ancestral state reconstrction on the maximum likelihood topology based on the combined dataset in Figure 3. Gains = losses = 1.0.

79 Eugymnanthea inquilna Calycella sy,ringa LovenellaOpercularella pumilalis Typical medusae Eucheilota . Medusoids Campanularia incksÜ Campanularia volubils Meconidia Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CA 1 Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopy.xis integra IT Obelia bidentata NC Obelia bidentata NS Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracils GB Clytia nolifòrmis Cfytia linearis BR Cfytia linearis IT Cfytia linearis NC Cfytia gracils MA Cfytia hemisphaerica Cfytia gracilis NC .. Cfytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomeiiea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Laomeaeaflexuosa FR Laomedeallexuosa IC Laomedeajlexuosa WS Laomedea inornata Obelia longissima AN Obelia longissima MA Obelia longissima NZ Obelia longissima WS

Figure 9. Ancestral state reconstrction on the maximum likelihood topology based on the combined dataset in Figure 3. Gains = 1.l - 1.4. Losses = 1.0.

80 Eugymnanthea inquilna CaTýce/la sy'ringa Opercularella pumila Typical medusae Lovenella gracilis Eucheilota bakeri Medusoids Campanularia hincksii Campanularia volubils Meconidia Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 l'ii~¡ig!!f1'JI Equivocal Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CAt Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopy.xis integra IC Obelia bidentata NC Obelia bidentata NS Clytia hummelincki SA Ctytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracilis GB Clytia noliformis Clytia linearis BR Clytia linearis IT Clytia linearis NC Ctytia gracilis WH Clytia hemisphaerica Clytia gracilis NC Clytia elsaoswaldae Ctytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomedea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Laomedea flexuosa FR Laomedeaflexuosa IC Laomedea flexuosa WS Laomedea'inornata Obelia longissima AN Obelia longissima WS Obelia longissima NA Obelia longissima MA

Figure 10. Ancestral state reconstrction on the maximum likelihood topology based on the combined dataset in Figure 3 Gains = 1.5. Losses = 1.0.

81 Eugymnanthea inquilna Calýcella sy'ringa Opercu/arella pumila Typical medusae Lovene/la gracilis Medusoids Eucheilota bakeri Campanu/aria hincksii Meconidia Campanu/aria vo/ubils Rhizocau/us verticillatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Itia!&ll equivocal Bonneviella sp. 3 Silcu/aria rosea Orthopyxis everta Orthopvxis integra IT Orthopyxis integra CAI Orthopyxis integra NZ Orthopyxis sargassico/a Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopy.xis integra IC Obelia bidentata NC Obelia bidentata NS C/vtia hummelincki SA Cfytia hummelincld IT Clytia pau/ensis Clvtia gracils IT Cfytia gracilis GB Clytia no/iformis Clytia linearis BR Clytia linearis IT Clytia linearis NC Clytia gracils MA Clytia riemisphaerica Clytia gracilis NC Clytia e/saoswa/dae Clytia sp. Gonothyraea /oveni ~ Gonothy.raea /oveni FR Laomedea ca/ceo/ifera Obe/a dichotoma Obelia genicu/ata FR Obelia genicu/ata NB Obelia genicu/ata JP Obelia genicu/ata NZ Laomedeaflexuosa FR Laomedea1lexuosa IC Laomedea flexuosa AS Laomedea'inornata Obelia /ongissima AN Obelia /ongissima WS Obelia /ongissima NZ Obelia /ongissima ~

Figure 11. Ancestral state reconstrction on the maximum likelihood topology based on the combined dataset in Figure 3. Gains = 1.6 - 1.9. Losses = 1.0.

82 Eugymnanthea inquilna Calycella sy,ringa Opercularella pumila Typical medusae Lovenella gracils Eucheilota bakeri Medusoids CampanuZaria hincksii Meconidia Campanularia volubils Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Iiw'E~~i~1 Equivocal Bonneviella sp. 3 Silcularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CAI Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopy.xis integra IC Obelia bidentata NC Obelia bidentata NS Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracils IT Clytia gracils GB Clytia noliformis Clytia linearis BR Clytia linearis IT Clytia linearis NC Clytia gracilis MA Clytia hemisphaerica Clytia gracilis NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomedea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Laomedea flexuosa FR Laomedeallexuosa IC Laomedea flexuosa WS Laomedea inornata Obelia longissima AN Obelia longissima WS Obelia longissima NZ Obelia longissima MA

Figure 12. Ancestral state reconstrction on the maximum likelihood topology based on the combined dataset in Figure 3. Gains 2: 2. Losses = 1.0.

83 Eugymnanthea inquilina Calycella sy'ringa Opercularella pumila Typical medusae Lovenella gracilis Medusoids Eucheilota bakeri Campanularia hincksii Meconidia Campanularia volubilis Rhizocaulus verticillatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia ¡'UiI)lsY,ll equivocal Bonneviella sp. 3 Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CA 1 Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopyxis integra IC Clytia hummelincki SA C(ytia hummelincki IT Clytia paulensis Clytia gracils IT Clytia gracilis GB Clytia noliformis Clytia linearis BR Clytia linearis IT Clytia linearis NC Clytia gracilis MA C(ytia hemisphaerica Clytia gracilis NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomeitea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia bidentata NC Obelia bidentata NS Laomedea flexuosa FR LaomedeaJ!exuosa IC LaomedeajJexuosa W A Laomedea inornata Obelia longissima AN Obelia longissima WS Obelia longissima NS Obelia longissima MA

Figure 13. Ancestral state reconstrction on the modified maximum likelihood topology. Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade. Gains = losses = l.O.

84 Eugymnanthea inqui/ina Calycella sy'ringa Opercularella pumila Typical medusae Lovenella gracilis Euchei/ota bakeri Medusoids Campanularia hincksii Meconidia Campanularia volubi/is Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 Silcularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CAl Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopyxis integra IC Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracils GB Clytia noliformis Clytia linearis BR .. Clytia linearis IT Clytia linearis NC Clytia gracils MA Clytia hemisphaerica Clytia g¡acilis NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomeâea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia bidentata NC Obelia bidentata NS La om edea flexuosa WS LaomedeaJlexuosa IC Laomedea flexuosa WS Laomedea. inornata Obelia longissima AN Obelia longissima WS Obelia longissima NZ Obelia longissima MA

Figure 14. Ancestral state reconstrction on the modified maximum likelihood topology. Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade. Gains = 1.1 - 1.9. Losses = 1.0.

85 Eugymnanthea inquilna Ca7ýcella sy'ringa Opercularella pumila Typical medusae Lovenella gracils Eucheilota bakeri Medusoids Campanularia hincksii Campanularia volubils Meconidia Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 equivocal Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CAI Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopyxis integra IC Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT "- Clytia gracils GB Clytia noliformis Clytia linearis BR Clytia linearis IT Clytia linearis NC Clytia gracilis MA Clytia nemisphaerica Clytia gracilis NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomeâea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia bidentata NC Obelia bidentata NS Laomedea flexuosa FR Laomedeaflexuosa IC Laomedea flexuosa WS Laomedea inornata Obelia longissima AN Obelia longissima WS Obelia longissima NZ Obelia longissima MA

Figure 15. Ancestral state reconstrction on the modified maximum likelihood topology. Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade. Gains 2: 2.0. Losses = 1.0.

86 Eugymnanthea inquilina Calycella sy'ringa Opercularella pumila Lovenella gracilis Typical medusae Eucheilota bakeri Medusoids Campanularia hincksii Campanularia volubils Meconidia Rhizocaulus verticilatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 I "l'!\iJII Equivocal Silcularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CAI Orthopyxis integra NZ Orthopyxis sargassicola .. Orthopyxis integra W A iI Orthopyxis integra CA2 Orthopyxis integra AK Orthopyxis integra IC Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracils GB Clytia noliformis Clytia linearis BR .. Clytia linearis IT Clytia linearis NC Clytia gracilis MA Clyfla hemisphaerica Clytia gracils NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomeâea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia bidentata NC Obelia bidentata NS Laomedea flexuosa FR Laomedeallexuosa IC Laomedea jJexuosa WS Laomedea inornata Obelia longissima AN Obelia longissima WS .. Obelia longissima NZ Obelia longissima MA

Figure 16. Ancestral state reconstrction on a modified maximum likelihood topology. Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade, and eampanularia hincksii is placed as the basal member ofthe eampanularia, Rhizocaulus, and Bonneviella clade. Gains = losses = 1.0.

87 Eugymnanthea inquilna Ca7ycella syringa _ Typical medusae/medusoids Opercularella pumi/a Lovenella gracilis Fixed gonophores Eucheilota bakeri Obelia medusae/meconidia Campanularia hincksii Campanularia volubi/is Equivocal Rhizocaulus verticilatus Bonneviella sp. 4 Bonneviella sp. 2 Bonneviella regia Bonneviella sp. 3 Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CA i Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopvxis integra CA2 Orthopyxis integra AK Orthopyxis integra IC Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracils GB Clytia nolirormis Clytia linèaris BR elytia linearis IT Clytia linearis NC Clytia gracilis MA Clytia hemisphaerica Clytia graciTs NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothy.raea loveni FR Laomedea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia õidentata NC Obelia bidentata NS Laomedea jlexuosa FR Laomedea Ilexuosa IC Laomedeajlexuosa WS Laomedea in ornata Obelia longissima AN Obelia longissima WS Obelia longissima NZ Obelia longissima WH

Figure 17. Ancestral state reconstrction on the modified maximum likelihood topology. Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade. Life cycles are placed in 3 categpri~s. Gains = losses = 1.0.

88 Eugymnanthea inquilina Calycella sy'ringa Opercularella pumila Typical medusae/medusoids Lovenella gracilis Fixed gonophores Eucheilota bakeri Campanularia hincksii Obelia medusae/meconidia Campanularia vol ubi/is Rhizocaulus verticillatus Bonneviella sp. 4 Bonneviella sp. 2 Bonneviella regia Bonneviella sp. 3 Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CA 1 Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopyxis integra IC Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracilis IT Clytia gracils OB Clytia nolifòrmis Clytia linearis BR Clytia linearis IT Clytia linearis NC Clytia gracils MA r'lytiaClytia gracilishemisphaerica NC lyt~a elsaoswaldae uonothyraealytlO sp. loveni ~ Gonothy.raea loveni FR Laomedea calceolifèra Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia bidentata NC belia bidentata NS Laomedea Ilexuosa FR Laomedeallexuosa IC Laomedeaflexuosa WS Laomedea inornata Obelia longissima AN Obelia longissÙna WS Obelia longissÙna NZ Obelia longissÙna MA

Figure 18. Ancestral state reconstrction on the modified maximum likelihood topology. Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade. Life cycles are placed in 3 categories. Gains 2: 1.1. Losses = 1.0.

89 90 Chapter 3 New taxonomic insights on the Campanulariidae (Cnidaria, Hydrozoa)*

*manuscript based on this chapter in preparation with Ferdinando Boero

Abstract Taxonomy is particularly challenging in the widely distributed hydrozoan family

Campanulariidae (Cnidaria, Leptomedusae) because of their complex life cycles and the diffculty in attrbuting morphological differences to genetic or environmental causes. Traditional morphological characters in the hydroids are based on colony form and theca characteristics. Nematocyst and hydranth features may be useful, but their utility is limited because they are time consuming to study and require live materiaL. Life cycle type is often used to define genera, but frequent life cycle transitions could result in paraphyletic classifications. DNA sequences have the potential to overcome many of the shortcomings of traditional hydrozoan taxonomy because they are relatively independent of environment and life cycle. Here, new taxonomic insights from a molecular phylogeny based on 2 nuclear (18S rDNA and calmodulin) and 2 mitochondrial (16S rDNA and cytochrome c I oxidase (COlD genes are discussed. Some aspects of traditional taxonomy are corroborated, while others are refuted. In particular, several species of Bonneviella, previously composing the family Bonneviellidae, are shown to be deeply nested in the Campanulariidae, and within the Obeliinae, multiple life cycle transitions have resulted in paraphyletic genera. The family and generic definitions are revised accordingly. Additionally, in some cases, nominal cosmopolitan species collected from multiple locations are suggested to represent cryptic species, while Obelia longissima may be truly cosmopolitan. Finally, new drawings of the obeliinid Laomedea (Gonothyraea) inornata are presented, showing that what were previously described as meconidia (medusoids) are really acrocysts (extracapsular development).

91 Introduction

The eampanulariidae- overview The Campanulariidae (Johnston, 1836) is a family ofhydrozoans (Cnidaria, Leptomedusae) that are widely distributed throughout the world. It comprises approximately 50+ species, including the well known Obelia, which is commonly used as a model hydrozoan in introductory biology courses. Following Cornelius (1982) the Campanulariidae are divided into 3 subfamilies: the Campanulariinae, which includes

Orthopyxis, Silcularia, eampanularia, and Rhizocaulus; the Clytiinae, which includes elytia; and the Obeliinae, which includes Obelia, Gonothyraea, Hartlaubella, and Laomedea. Within each subfamily, genera are defined in part on the basis of life cycle, or gonophore, type. The purpose of this paper is to review campanulariid taxonomy and incorporate the molecular results of Chapter 2. Revisions of the Campanulariidae and the Obeliinae based on their strongly supported findings are presented.

How many species? The number of described campanulariid species is difficult to calculate because some nominal taxa may represent cryptic species complexes, while others may not be valid. The most recent revisions of the Campanulariidae were by Cornelius (1975; 1982; 1990). His revisions created considerable debate among the hydrozoan community because of his extensive synonymizations, particularly for Obelia. Among the Obelia, he merged 120 nominal species into 3 (Cornelius 1975), although he later increased that number to 4 (Cornelius, 1990). Other authors recognize additional species (e.g., Stepanjants, 1998; Kubota 1999; Bouilon and Boero, 2000).

Cornelius's (1982) revision of the entire family dealt primarily with those found in the eastern North Atlantic, and the other campanulariid genera have not had a worldwide revision similar to that of Obelia (1975). Nominal taxa in these groups may not all represent valid species. In particular, some elytia are known only from the hydroid or medusa stage, and elucidation of their complete life cycles wil be important for a future revision (Bouilon and Boero 2000).

92 Species definitions in the Campanulariidae are primarily based on morphological differences (e.g., Cornelius, 1982), rather than breeding experiments, which would test the biological species concept. Breeding experiments are not feasible for most campanulariids as many, such as Obelia, are very diffcult to keep in laboratory culture. Also, such experiments obviously require live matenal. Alternatively, lineage-based species concepts, such as the phylogenetic species concept, identify monophyletic clades (Sites and Crandall, 1997). Monophyletic patterns are often based on, but not limited to, mitochondrial DNA markers. The concordance concept looks for concordant patterns of monophyly using multiple independent markers (Avise, 2000). Lineage-based species concepts have not been applied to the Campanulariidae, however.

Taxonomic characters Overall colony form and characteristics of the hydrotheca are important taxonomic characters in the Campanulariidae. These characteristics include whether the hydroid colony is reptant (creeping) or erect, the degree of branching, whether the stem is mono- or polysiphonic, hydrothecal dimensions, the type of hydrothecal margin, and the presence/absence of a subhydrothecal spherule (spherical annulation). These characters are useful because they remain intact in preserved specimens. However, they may also exhibit considerable phenotypic plasticity (Ralph, 1956; Cornelius, 1982). The degree to which differences in gross morphology are due to environmental or genetic causes is unkown, and this presents a major obstacle to their taxonomic use. Morphological characteristics of the nematocysts, or stinging capsules, are taxonomically useful in many groups of hydro ids (Gravier-Bonnet, 1987). Östman (1979; Östman 1982, Östman, 1983; Östman et aI., 1987; Östman, 1987) has characterized nematocysts of Campanulariid hydroids, and found species-specific differences. However, the degree of phenotyic plasticity in nematocysts is unkown. In their comparison of conspecific Swedish and Italian Campanulariids, Östman et al. (1987) found size differences between nematocysts from northern and southern specimens, suggesting that some nematocyst characters can be plastic.

93 Hydranth characteristics may also be distinctive; for example, a peduncled (flared or trmpet-shaped), rather than conical, hypostome distinguishes campanulariids from other thecate hydroids. Frustrated with limitations of traditional taxonomic methods, Cornelius (1987a; 1987b) proposed using additional characters from live hydranths. In a preliminary survey of some European hydroids, including some campanulariids, he found that hydranth characters may be less variable than hydrothecal characters. A critical drawback for this approach is that live hydroids must be examined, because, when thecate hydroids are preserved, their hydranths contract into the hydro theca, rendering them unavailable for study. Östman (1982; 1983) examined differences in isoenzymes from Scandinavian campanulariids. She found inter- and intraspecific gel band differences for the enzyme acid phosphatase, and suggested that taken with other characters, isoenzyme banding patterns could be taxonomically usefuL. However, no further work since then has been done with isoenzymes.

Gonophore type as a generic character An ongoing debate in hydrozoan systematics is whether to use gonophore (sexual stage) development as a generic character. Within families, hydro ids bearing liberable medusae, medusoids, or fixed gonophores are frequently placed in separate genera. The use of gonophore type to define genera has been criticized because if gonophore transitions happen frequently, paraphyletic genera result (e.g., in the Tubulariidae (Petersen, 1990) and the Hydractiniidae (Cunningham and Buss, 1993)). Curent classifications of the Campanulariidae use gonophore tye, in part to define genera, although this approach has been recently questioned by Boero et al. (1996). eampanulariid phylogeny Molecular genetic data have the potential to clarify both campanulariid taxonomy and evolutionary relationships, because markers relatively independent of life cycle and environment are available. Govindarajan (Chapter 2) created a molecular phylogeny of

94 the Campanulariidae based on 2 nuclear genes (18S rDNA and calmodulin) and 2 mitochondrial genes (16S rDNA and cytochrome c oxidase subunit I (COlD. Their results corroborate some aspects of morphological taxonomy but clearly refute others, indicating that the Campanularidae, and possibly the entire Leptomedusae, need revision. Their results are summarized in Figues 1 and 2. Figure 1 shows the combined analysis of all 4 genes (corresponding to Figure 3 in Chapter 2), and Figure 2 shows the gonophore type mapped on to a bootstrap consensus tree and the ancestral character states reconstructed using parsimony (corresponding to Figure 14 in Chapter 2). Details of the analyses and figures of the data analyzed individually are in Chapter 2

Molecular results

Family eampanulariidae The family Campanulariidae plus Bonneviella spp. and exclusive of Bilardia falls out as a monophyletic group, although it is not well supported by bootstrap analysis (Figure 1). The Campanulariidae appears to be closely related to Eucheilota bakeri

(Eucheilotidae) and Lovenella gracils (Lovenellidae). Interestingly, the hydroid of Eucheilota bakeri was originally placed in the Campanulariidae, as elytia bakeri (Torrey, 1904). However, the medusa displays characters typical ofthe Eucheilotidae (with lateral cirri), where it was placed by Mayer (1910) and Kramp (1961). Despite their placement in different families, the hydroids of Eucheilota bakeri, found only in southern California and Mexico (Fraser, 1946), and Lovenella gracils, found only in the southeastern USA (Calder, 1971), display many morphological and ecological similarities. Both live on bivalves inhabiting sandy beaches, which is an unusual habitat for hydroids. Morphologically, they are similar, although Lovenella gracilis hydroids have an operculum (covering of the hydranth opening), while Eucheilota bakeri hydroids, like campanulariid hydroids, do not (Calder, 1971). In addition to their morphological and ecological similarities, the relatively little sequence divergence between the two could suggest they are sister taxa, perhaps separated by the Isthmus of Panama. Further study with additionalleptomedusan taxa is necessary to

95 understand the relationship between Eucheilota bakeri and Lovenella gracils, and their relationship with the Campanulariidae.

Familyeampanulariidae. the Bonneviellidae Several species of Bonneviella (Bonneviellidae) which were originally designated as outgroups (Chapter 2), fall inside the Campanulariidae (Figure 1). The Bonneviellidae Broch, 1909 is composed of 8 species belonging to the genus Bonneviella. All are found in the North Pacific, and one species, B. grandis, is also found in the North Atlantic (Nutting, 1915; Naumov, 1960). All possess fixed gonophores rather than medusae or medusoids. The Bonnevielldae is traditionally distinguished from the Campanulariidae by the absence of a peduncled hypostome and the presence of a preoral gastric cavity (Nutting, 1915). Nutting (1915) agreed with Broch (1909) that Bonneviella possess a "veloid", or membrane, that divides the gastrc cavity. Nutting (1915) further suggested that they have no real proboscis (hypostome), but rather the oral surface is a depression whose lowest point is the mouth. Naumov (1960), however, pointed out that the veloid is actually a tyical conical hypostome, and it is actually the tentacle bases projecting into the gastric cavity that divide it. Bouilon (1985), placing the Bonneviellidae in the order

Proboscoida Broch, 1909 and superfamily Campanulariidea Bouilon, 1984, suggested affnities with the Campanulariidae and the Lafoeidae. The molecular phylogeny strongly places 4 Bonneviella species deeply nested in the Campanu1ariidae, in the Campanulariinae (Figue 1). Their inclusion indicates that the peduncled hypostome is not a campanulariid synapomorphy, as long believed

(Nutting, 1915). Bonneviella appears to be closely related to eampanularia volubils and Rhizocaulus verticilatus, which were also collected in the North Pacific.

Familyeampanulariidae. Bilardia The genus Billardia was first identified and placed in the Campanulariidae by Totton (1930). Currently, it is composed of 3 species found only in the southern hemisphere: Bilardia intermedia (Blanco, 1967), B. novaezealandiae (Totton, 1930), and

96 B. subrufa (Jäderholm, 1905). Milard (1975) suggested Bilardia may have affinities with the Lafoeidae, based on their curved bilaterally symmetrical hydrothecae, or the Syntheciidae, based on their occasionally slightly adnate (side partially in contact with stem) hydrothecae. Cornelius (1982) pointed out that if, as Totton (1930) suggested, the blastostyles (gonophores) were produced in place of hydranths, within the hydrothecae, then Bilardia belongs in the Syntheciidae sensu Milard (1975) rather than the Campanulariidae. The sertulariid genus Fraseroscyphus also has gonophores in place of hydrothecae (Boero and Bouilon, 1993). However, Bilardia has been placed in the Campanulariidae by Ralph (1957), Blanco (1967), Vervoort (1972), and most recently, Vervoort and Watson (2003). The molecular phylogeny, however, (Figure 2) strongly indicates that Bilardia does not belong in the Campanulariidae, although additional analyses are necessary to examine its placement in the Lafoeidae or Syntheciidae.

The eampanulariinae The Campanulariinae, including Bonneviella, is a strongly supported clade (Figure 1 ).Within the Campanulariinae clade, many relationships are not that well supported and additional generic markers will probably be necessary to make definitive conclusions. The Bonneviella species form a monophyletic clade, although it is not supported by bootstrap analysis. However, they appear to be closely related to two other species collected in the North Pacific, eampanularia volubils and Rhizocaulus verticillatus (Figure 1). Although the position of Bonneviella within the Campanulariidae was unexpected, Rees and Thursfield (1967) noted similarities between eampanularia volubils and Rhizocaulus verticilatus. They speculated that eampanularia volubils and Rhizocaulus verticillatus might actually be conspecific, although Cornelius (1982) disagreed. While a close relationship between the two is evidenced by the molecular phylogeny, they do not fall out together as would be expected if they were conspecific. The frequency of medusoid reduction in the Campanulariinae cannot be determined from the molecular phylogeny because of inadequate phylogenetic resolution,

97 uncertainties in assumptions of ancestral state reconstruction, and possible bias in taxon sampling (Chapter 2). While most fixed gonophore-bearing species fall together in a monophyletic clade (Bonneviella, eampanularia volubils, Rhizocaulus verticiilatus), the apparent monophyly may represent a single, geographically restricted radiation. Members of this clade were all collected in the North Pacific, while eampanularia hincksii, the only other fixed gonophore-bearing species included in the phylogeny, comes from the Mediterranean, and falls outside this clade (although its position is not well supported). eampanularia species from other parts of the world (e.g., Millard, 1975) could not be obtained for this study and additional sampling on a global scale is necessary. Orthopyxis integra is considered to be an exceptionally morphologically variable, cosmopolitan species (Cornelius, 1982). The molecular results suggest that 0. integra could be a complex of cryptic species. O. integra from several locations do not form a monophyletic clade, although specimens from some locations in the North Pacific and North Atlantic appear to fall out together, as do 0. integra from New Zealand and California (Figure 1).0. integra from Italy (formerly 0. asymmetrica) falls outside this clade and appears allied with another Italian Orthopyxis, 0. everta. A second form of 0. integra from Italy, which grows on a variety of substrates as opposed to only on the seagrass Posidonia like the 0. asymmetrica form, was not examined. More samples, especially multiple samples from within locations, are necessary to determine whether the 0. integra clade represents one or a group of closely related species. Species boundaries should be examined by looking for patterns of reciprocal monophyly with molecular data (e.g., Govindarajan et aI., submitted; Chapter 4).

The Obeliinae and elytiinae A close relationship between the Clytiinae and Obeliinae is suggested, although the arrangement of the clades does not have much bootstrap support (Figure 1). This is consistent with the hypothesis ofBoero et al. (1996) and in contrast to Cornelius (1982). The implications of this relationship for life cycle evolution are discussed in Chapter 2 and partially summarized in Figure 3. elytia is monophyletic (but note low bootstrap

98 support for the position of elytia hummelincki), although appears nested within the Obeliinae due to the basal position of Obelia bidentata in the maximum likelihood topology (tree) (Figure 1). However, parsimony analysis places Obelia bidentata in the basal position of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade (Figure 1; Figure 3). Obelia bidentata DNA sequences appear to have very long branch lengths, indicating a lot of change, and making it diffcult to ascertain their position in the phylogeny. Additional markers wil be necessary to determine the position of Obelia bidentata with certainty. Within the elytia clade, elytia hummelincki is basal (again noting this position has low bootstrap support). elytia hummelincki possesses a typical elytia medusa, although its hydroid has a subhydrothecal spherule (spherical thickening underneath hydranth), typical of most Campanulariinae. The ancestral Campanulariid may have had a form like,Clytia hummelincki, with one descendent lineage becoming the Campanulariinae, and the other the Clytiinae + Obeliinae. However, the molecular results do not provide much support for the position of elytia hummelincki and the relationship between the elytia, Obeliinae and Campanulariinae lineages, so this hypothesis remains to be tested. elytia gracils collected from different localities do not form a monophyletic clade, indicating that it, like Orthopyxis integra, likely represents several cryptic species (Figure 1). Interestingly, Cornelius (1982) synonomized elytia gracilis with the cosmopolitan elytia hemisphaerica, but subsequently elytia gracils was shown to be distinct on the basis ofnematocysts by Östman (summarized in Östman, 1987). In a detailed morphological and life cycle study, Lindner et al. (in prep) showed that a Brazilian "elytia gracilis" was distinct and resurrected the name elytia elsaoswaldae for it. Additional investigations of samples from throughout its global range are necessary to assess its species-level diversity. There are two main Obeliinae clades. The first includes Gonothyraea loveni, Obelia dichotoma, Obelia geniculata, and Laomedea calceolifera. The second clade includes Obelia longissima, Laomedea flexuosa, and Laomedea inornata. Obelia

99 bidentata has a very long branch length, indicating a lot of change, but may be allied with the second clade (see above; and also based on the L8S data alone (Chapter 2)). There is some morphological and physiological evidence supporting the existence of the two Obeliinae clades. First, the medusae of Obelia geniculata and 0. dichotoma are identical, and specimens identified from the medusa stage as Obelia lucifera are thought to consist of both O. geniculata and 0. dichotoma individuals (Cornelius, 1975). Obelia longissima medusae may differ slightly from nominal 0. lucifera medusae (Cornelius, 1990; Kubota 1999). Secondly, Miler (1973) found that gametes from Gonothyraea loveni and Laomedea calceolifera were attracted to each other, but that gametes from Laomedea calceolifera and Laomedea flexuosa were not attracted to each other. The phylogeny suggests that Obelia medusae were likely reduced multiple times (Figure 3). The alternative, that medusae were reduced only once, requires multiple independent origins of Obelia medusae and so seems less probable if it is assumed that gains in structure are even slightly less likely than losses (Chapter 2). In any case, the obeliinid genera are clearly not monophyletic. Therefore, these results indicate that the taxonomic practice of using gonophore type to define genera is not appropriate in the Obeliinae.

Obelia longissima - a true cosmopolite? Cornelius (l975) synonymized Obelia longissima, as well as many other nominal Obelia species, into Obelia dichotoma. Based on nematocyst differences and electrophoretic patterns, Östman subsequently (1979, 1982a,b, 1983 a,b) showed that 0. longissima was distinct, and this was recognized by Cornelius (1990; 1995). The molecular results confir that Obelia longissima and Obelia dichotoma are distinct; indeed, they fall in different Obeliinae lineages. 0. longissima specimens from the North Atlantic (MA, USA and Iceland), the White Sea, the Antarctic peninsula, and New Zealand were included in the molecular phylogeny (Figure 1). The results indicate a highly supported monophyletic clade with very little sequence divergence in all 4 genes from these widely separated localities,

100 consistent with what would be expected for a cosmopolitan species. Obelia longissima has the potential for long distance dispersal via its hydro ids (rafting on algae) and planktonic medusae, although such high dispersal potential may not always be realized (Govindarajan et aI., submitted; Chapter 4). An alternative possibility is that the global distribution of Obelia longissima might be the result of several anthropogenic introductions, but it should be noted that one of the locations (off of the Antarctic peninsula, Govindarajan et al. in prep) is not near a major port. In contrast, comparison of mitochondrial genes of Obelia geniculata from the North Atlantic, North Pacific (Japan), and South Pacific (New Zealand) suggest that 0. geniculata is composed of at least 3 crytic species (Govindarajan et aI., submitted;

Chapter 4). Surprisingly, of the all the synonomizations in Cornelius (1975),0. geniculata generated the least controversy (Cornelius, 1990). Thus, ofthe 4 species of Obelia recognized by Cornelius (1990; 1995), some, like 0. longissima, may be trly cosmopolitan, while others, like 0. geniculata, appear to represent multiple cryptic species. In particular, it will be interesting to compare 0. dichotoma specimens from multiple localities, as this species is the most variable and has the most synonyms.

Laomedea inornata and acrocysts The Obeliinid Gonothyraea inornata Nutting 1901 was first discovered in Alaska, and later found in China (Hargitt, 1927), Washington (Fraser, 1937) and Argentina (Blanco, 1968). It was placed in the genus Gonothyraea because the gonosome has an acrocyst (external capsule on top of the gonotheca), which was thought to be similar to meconidia, although it possesses no medusoid featues (Nutting, 1915). Inspection of fertile colonies obtained from a floating dock in Friday Harbor, W A confirmed that this strcture is a simple acrocyst (extracapsular development; Figure 4) and it is referred to as Laomedea in ornata in Chapter 2 and Figue 1. This species is similar to Laomedea neglecta , which is found in the North Atlantic. L. neglecta also posesses an acrocyst, but differs from L. inornata in that the hydrothecal margin is toothed, although it sometimes abrades (Cornelius, 1982). Also L.

101 neglecta acrocysts contain relatively few ova (3 reported by Wright, 1863), while L. inornata can contain more (6-8 observed; Figure 3). The acrocyst has been postulated to be an intermediate stage in a series of gonophore reduction, between Gonothyraea and other Laomedea and Hartlaubella (Cornelius, 1982). Molecular analysis indicates that there is neither a straightforward series of gonophore reductions nor any affinities between the acrocyst-bearing L. inornata with the meconidia-bearing Gonothyraea. Rather, L. inornata appears to be the direct result of a medusa reduction of Obelia longissima. Additional studies are necessary to determine if L. inornata and L. neglecta are closely related and the acrocyst evolved only once, or if L. inornata and L. neglecta are relatively distant and the acrocyst evolved multiple times. Given that medusa reduction apparently occurred multiple times in the Obeliinae, a single origin for the acrocyst should not be assumed. eampanulariid revision Here, we present a revision of the Campanulariidae, in light of the molecular phylogeny. The diagnoses are modified from Cornelius (1982) and Bouilon and Boero (2000) to incorporate the new findings. This entailed two major changes which were highly supported: (1) inclusion of the Bonneviellidae, as the genus Bonneviella in the Campanulariinae; and (2) merging the Obeliinae genera (Gonothyraea, Laomedea,

Obelia, and Hartlaubella) into a single genus, Obelia. Subfamily and generic definitions from Cornelius (1982) and Bouilon and Boero (2000) that were not changed are not presented.

Family Campanulariidae Johnston, 1836 Diagnosis Medusa: (From Bouillon and Boero, 2000) When present, Leptomedusae with short manubrium; without gastric peduncle; typically with 4 radial canals but sometimes more; with or without velum; with gonads on radial canals, completely surrounding them and separated from the manubrium; hollow or solid tentacles; with or without tenon-like

102 rudimentary bulbs; without marginal cirri; without excretory papillae or pores; numerous (8-200) closed velar marginal statocysts; no ocelli.

Hydroid: (Modified from Cornelius, 1982) Hydroids forming erect or stolonal colonies; hydrothecae bell-shaped or campanulate, radially or secondarily bilaterally symmetrical; pedicellate, rim cusped or not, lacking operculum, with basal diaphragm or inward annular projection of perisarc; nematophores absent, hydranth when known generally tubular with flared or globose hypostome delimiting a "buccal cavity", with one whorl of filiform tentacles, gastric endoderm of uniform strcture; hydrothecal spherules present or not; with free medusae, medusoids, or sporosacs.

Remarks The medusa diagnosis is not changed from Bouilon and Boero (2000). The hydroid diagnosis is modified to include the Bonneviellidae, which possess conical, rather than peduncled, hypostomes and have a pre-oral gastric cavity. The removal of Bilardia from the Campanulariidae does not require a change in diagnosis. The molecular phylogeny (Figure l) also showed a close relationship with Eucheilota bakeri and Lovenella gracilis. Their potential placement inside the Campanulariidae cannot be ruled out at this time and should be further investigated in the future.

Subfamily Campanulariinae

Diagnosis: Campanulariid hydro ids with reptant or branching colonies. Stem usually monosiphonic but polysiphonic in Rhizocaulus and some Bonneviella. Subhydrothecal spherule usually present. No tre hydrothecal diaphragm. Hypostome usually peduncled but conical in Bonneviella. Pre-oral gastric cavity in Bonneviella. With medusoids that mayor may not be released, or fixed gonophores.

103 Genus Bonneviella Broch, 1909

Diagnosis: Campanulariinid hydro ids with conical hypostome and the tentacle bases projecting into the gastric cavity, forming a pre-oral gastric cavity (Naumov, 1960). Subhydrothecal spherule may not be present. With fixed gonophores.

Remarks: If further molecular and morphological investigation indicate that medusoid reduction in the Campanulariinae occurred multiple times, the Bonneviella and other campanulariinid genera be combined into a single genus, such as in the Obeliinae (below). At present, however, insuffcient taxon sampling and resolution in the molecular phylogeny preclude such a change. With the exception of eampanularia hincksii, the molecular phylogeny places the fixed-gonophore bearing Campanulariinae in a single monophyletic clade. However, members of this clade are all ofa North Pacific origin, and so may be the result of a single radiation. The position of eampanularia hincksii, obtained from Italy, is not well supported. Additional sampling on a global basis and possibly the use of new markers are necessary to determine the frequency of medusoid reduction in the Campanulariinae.

Subfamily Obeliinae

Diagnosis: Campanulariid hydro ids with erect hydrocauli forming branched or unbranched, mono- or polysiphonic colonies; true hydrothecal diaphragm; no sub- hyrothecal spherule; stolon not anastomosing; with liberable medusae, medusoids, or fixed gonophores. Campanulariid medusae without velum and with solid tentacles.

Remarks. The diagnosis combines the definitions in Cornelius (1982) and Bouilon and Boero (2000). No new modifications are presented here. The molecular phylogeny suggests a close relationship with elytia, consistent with the scheme presented by Boero et ai. (1996), rather than as independent lineages suggested by Cornelius (1982). Indeed, the molecular phylogeny suggests that the Obeliinae and Clytiinae may be paraphyletic

104 with respect to each other; however, the arrangement of their respective lineages is not well resolved. Therefore, no changes at the subfamily level are undertaken here.

Genus Obelia Peron and Lesueur, 1810

Obelia Peron and Lesueur, 1810

Gonothyraea Allman, 1864

Hartlaubella Poche, 1914

Laomedea Lamouroux, 1812

Diagnosis Hydroid - Colonies erect, branching or unbranching; stolon not anastomosing; hydrocaulus simple or polysiphonic; subhydrothecal spherule always absent; perisarc thickness variable; hydrothecal margin variable (smooth, wavy, or toothed); radially symmetric; hydranth fully retractable into hydrotheca.

Medusae/sexual stage - Medusae, meconidia, or fixed sporosacs, occasionally with acrocysts. Medusae when present with solid tentacles; 16 or more tentacles present upon release; velum absent; flat; one quadrangular manubrium with 4 lips; 4 radial canals; no lateral cirri; and 8 marginal statocysts.

Remarks Cornelius (1999) justifies the existence of a separate genus for Hartlaubella also because of its polysiphonic stem. However, older Obelia bidentata colonies have polysiphonic stems as well as occasionally Obelia dichotoma and Obelia longissima. Gonothyraea is separated from Obelia solely by its meconidia, while Hartlaubella is distinguished by its fixed gonophores, and also by its polysiphonic stem.

Acknowledgements

105 We are grateful to numerous people, including A. Collns, J. Coyer, C. Gravil, B. Grossman, A. Hart, L. Henry, Y. Hirano, E. Horgan, A. Lindner, i. Kosevich, S. Kubota, M. P. Miglietta, S. Piraino, K. Reise, P Schuchert and N. Trowbridge for collection assistance and/or providing samples for the molecular analysis. C. Gravili provided assistance with the drawings. C. Cunningham, K. Halanych, and L. Madin provided valuable advice throughout the duration of this project. A. Govindarajan was supported by WHOI Academic Programs and an NSF PEET grant (DEB--9978131) to C. Cunningham. Additional research funds were provided by WHOI Ocean Ventures Fund, Society for Integrative and Comparative Biology, and the MIT-Italy club.

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111 Figure 1. Maximum likelihood phylogeny from a heuristic search based on the combined 18S rDNA, calmodulin, 16S rDNA, and COI sequences (Figure 3 in Chapter 2). First number refers to maximum likelihood bootstrap, second number refers to parsimony bootstrap, and third number refers to Bayesian posterior probability. In contrast to the likelihood topology shown here, parsimony analyses placed Obelia bidentata as the basal member of the clade with Laomedea flexuosa, Laomedea inornata, and Obelia longissima (parsimony bootstrap value = 79). The 18S rDNA sequences alone also strongly support this position for Obelia bidentata (Figure 4 in Chapter 2). Subfamily lineages are indicated. Location codes: AN=Antarctic peninsula; AS=Aleutians; BR=Brazil; CA=California; FR=France; GB=Georges Bank; IC=Iceland; IT=Italy; JP=Japan; MA=Massachusetts; NB=New Brunswick; NJ=New Jersey; NZ=New Zealand; NC=North Carolina; NS=North Sea; SA=South Africa; W A=Washington. Gonophore tye indicated in bold after the taxon name. T = typical medusae; D = medusoids; F = fixed gonophores; M = meconidia; 0 = Obelia medusae.

112 II 00 Eugymnanthea inquilina D Calycella syringa F 98i78/100 Opercularella pumila F 100/99/100 Lovenella gracilis T Eucheilota bakeri T Campanularia hincksiiF Campanularia volubils F 100/1 00/100 Rhizocaulus verticilatus F Bonneviella sp. 4 F Bonneviella sp. 2 F Bonneviella regia F Campanulariinae 52//96 Bonneviella sp. 3 F (+ Bonnevielldae) Silicularia rosea D 96/ II 100/99/100 Orthopyxis everta D Orthopyxis integra IT D 53//93 Orthopyxis integra CA1 D Orthopyxis integra NZ D //56 OrthoP.v~ris sargassicola D Orthopyxis integra WAD Orthopyxis integra CA2 D Orthopyxis integra AK D Orthopyxis integra IC D 59//88 100/100/100 .Ob~lia bidentata NC 0 I Obeliinae Obelia bidentata NS 0 100/1 00/1 00 Clytia hummelincki SA T qvtia huiimelincki IT T Clytia paulensis T /65/8 //80 Clytia gracils IT T Clytia gracilis GB T 96/80/100 97/55/100 Clytia noliformis T 100/ I 0011 00 Clytia linearis BR T Cl" 84/65/100 Clytia linearis IT T ytnnae 100/1001100 Clytia linearis NC T 91152/100 ClyÛa gracilis MA T ClyÛa hemisphaerica T /53/59 ClyÛa gracilis NC T Clytia elsaoswaldae T 99/100/100 Clytia sp. T Gonothvraea loveni MA M Gonothyraéa loveni FR M Laomedea calceolijèra F . . Obelia dichotoma 0 100/10011e9elia geniculata FR 0 Obelia geniculata NB 0 //61 Obelia geniculata JP 0 Obelia geniciilata NZ 0 Obeliinae 100/1 00/1 00 Laomedeajlexuosa FR F Laomedeajlexuosa IC F 10011 00/ I 00 100/100/100 Laomedeaflexuosa WS F Laomedea inomata .F Obelia longissima AN 0 99/100/100 Obelia longissima WS 0 84/96/89 Obelia longissima IC 0 Obelia longissima NZ 0 Obelia longissima MA 0 - 0.01 substitutions/site

113 Cholorohydra viridissima Hydra circumcincta Bilardia siihriifa Stegella lobata Melicertissa sp. 100/1 00/1 00 Gymnangium lians Selaginopsis comigera CaZvcella syringa Opercularella pumila 170/100 Tiaropsidium kelsyi Eugymnanthea inquilna Blackrordia virginica 100/1 00/1 00 Aequorea aequorea Aeqorea victoria Sileiilaria rosea 95/100/100 Orthopyxis illtegra - AK

611/ Callpaliiilaria98/941) 00 . lii1ieksii Ort IopyXIS everta /601 Callpaliiilaria VOliihils 58110 Bonneviella regia 0/85/1 0 Rhizoeaiiliis verticillatiis 100/97/100 66/163 Bonneviella sp. 2 99/1 1100 Lovenella gracilis Eucheilota bakeri Goiiotliyraea loveiii - MA ,015i100 /67/10094, Obelia 4/10l6helia.geiiieiilata diehotoma - FR

Laolledea ealeeolifera

162/100 Obelia bide1tata - NC /751100 Laomedea flexiiosa - FR 100/100/100 Obelia loligissilla - MA 93/100/100 Laomedea inornata Clytia hllllmelineki - IT Clytia paiilensis

Clytia sp. Clyia liiiearis Clytia gracilis - GB Clytia hemisphaeriea - 0.01 substitutions/site

Figure 2. Maximum likelihood phylogeny of the Campanulariidae rooted with additional Leptomedusae and Hydridae sequences (Figure 2 in Chapter 2). First number refers to maximum likelihood bootstrap, second number refers to parsimony bootstrap, and third number refers to Bayesian posterior probability. Location codes as in Figue 1. Note Bilardia falls well outside the primary campanulariid clade.

114 Eugymnanthea inquilna Calycella sy'ringa Opercularella pumila Typical medusae Lovenella gracilis Medusoids Eucheilota bakeri Campanularia hincksii Meconidia Campanularia volubilis Rhizocaulus verticillatus Fixed gonophores Bonneviella sp. 4 Bonneviella sp. 2 Obelia medusae Bonneviella regia Bonneviella sp. 3 Silicularia rosea Orthopyxis everta Orthopyxis integra IT Orthopyxis integra CAI Orthopyxis integra NZ Orthopyxis sargassicola Orthopyxis integra W A Orthopyxis integra CA2 Orthopyxis integra AK Orthopyxis integra ie Clytia hummelincki SA Clytia hummelincki IT Clytia paulensis Clytia gracils IT Clytia gracils GB Clytia noliformis Clytia linearis BR Clytia linearis IT Clytia linearis NC Clytia gracilis MA Clytia hemisphaerica Clytia gracilis NC Clytia elsaoswaldae Clytia sp. Gonothyraea loveni MA Gonothyraea loveni FR Laomedea calceolifera Obelia dichotoma Obelia geniculata FR Obelia geniculata NB Obelia geniculata JP Obelia geniculata NZ Obelia bidentata NC Obelia bidentata NS Laomedea flexuosa WS Laomedeaflexuosa IC Laomedeajlexuosa WS Laomedea inornata Obelia longissima AN Obelia longissima WS Obelia longissima NZ Obelia longissima MA

Figure 3. Ancestral state reconstrction on the modified maximum likelihood topology (Figure 14 in Chapter 2). Obelia bidentata is placed as the basal member of the Laomedea flexuosa, Laomedea inornata, and Obelia longissima clade. Gains = 1.1 - 1.9. Losses = 1.0.

115 A. B.

r'~) jI //

) \\\ I O.lmm

acrocyst c. ~ /~¡ ( \ (J'~~~\ ~ \" .' .\ \~~-, i \\\,1\ / , )/'

\ '. ~'ì ....1, 1\\\ '2£'$! k--~H lmm I 0. mm1 \ ,~. \-'v) ì~.=j i lmm

Figure 4. Laomedea inornata. A. colony branch. B. hydranth. C. gonophores, showing egg and planulae development and the acrocyst.

116 Chapter 4 Mitochondrial evolution and phylogeography in the hydrozoan Obelia geniculata (Cnidaria)*

* manuscript based on this chapter coauthored with Ken Halanych and Cliff Cunningham has been accepted for publication in Marine Biology

Abstract

The distribution and genetic strcture of many marine invertebrates in the North Atlantic have been influenced by the Pleistocene glaciation, which caused local extinctions followed by recolonization in warmer periods. Mitochondrial DNA markers are typically used to reconstruct species histories. Here, two mitochondrial markers (16S rDNA and cytochrome c oxidase I (COlD were used to study the evolution ofthe widely- distributed hydrozoan Obelia geniculata (Linnaeus, 1758) from the North Atlantic and

the Pacific, and more specifically in the context of the North Atlantic phylogeography. Samples were collected from six geographic localities between 1998-2002. Hydroids from the North Atlantic, North Pacific (Japan), and South Pacific (New Zealand) are

reciprocally-monophyletic and may represent cryptic species. Using portions of the L6S

rDNA and COI genes and the date of the last trans-Arctic interchange (3.1 -4.1 mya), the first calibrated rate of nucleotide substitutions in hydrozoans is presented. Whereas extremely low substitution rates have been reported in other cnidarians, mainly based on anthozoans, substitution rates in 0. geniculata are comparable to other invertebrates. Despite a life history that ostensibly permits substantial dispersal, there is apparently considerable genetic differentiation in 0. geniculata. Divergence estimates and the

presence of unique haplotyes provide evidence for glacial refugia in Iceland and New Brunswick, Canada. A population in Massachusetts, USA appears to represent a relatively recent colonization event.

117 Introduction The rate of mitochondrial nucleotide substitution in Cnidaria, particularly in the Anthozoa, is thought to be considerably lower than in other marine invertebrates (Romano and Palumbi, 1997; Medina et aI., 1999; Van Oppen et aI., 1999) and may be 10 to 20 times lower than in vertebrates (Shearer et aI., 2002). Cnidarian mitochondral evolution has been best studied in the Anthozoa, because there is a fossil record against which sequence divergence can be calibrated. Generalizations from these studies may not be applicable to other cnidarian clades. Analysis of the scyphozoan Aurelia aurita COI suggests that the substitution rate may be higher in this group (Dawson and Jacobs, 2001). The only previous study on a hydrozoan (Shearer et aI., 2002) used a relative rate test on COI amino acid sequences, and found that the hydrozoan (Limnomedusa) Maeotias sp. was evolving significantly more slowly than echinoderms, molluscs, and arthropods. A variety of mechanisms, such as selection, a recent bottleneck, introgression, and mismatch repair, could contribute to this unusually slow rate (Shearer et aI., 2002). Paricularly interesting is the possibility of a mismatch repair system. Mitochondrial gene content is highly conserved among metazoans, but some anthozoans (e.g., octocorals) possess a gene coding for a mismatch repair protein (MSH) that has not been found in any other metazoan mitochondral genome (Pont-Kingdon et aI., 1995, Pont-Kingdon et aI., 1998; France and Hoover, 2001). However, it is not known if and how this gene functions in mismatch repair in anthozoan mitochondria (Pont-Kingdon et aI., 1998; Shearer et aI., 2002). Biogeographic events can sometimes provide firm upper and lower bounds for divergence when a fossil record is not available (e.g., Cunningham et aI., 1991; Knowlton and Weigt, 1998; Wares and Cunningham, 2001). Here, assuming migration through the trans-Arctic interchange (Vermeij, 1991), we provide the first calibrated substitution rates for a hydrozoan, Obelia geniculata. We then use our calibrated substitution rates to interpret the timing of phylogeographic events for 0. geniculata in the North Atlantic.

118 The hydrozoan Obelia geniculata (Leptomedusa, Campanulariidae) is very widely distributed (Cornelius, 1975). It is found on both sides of the north and south Atlantic and Pacific Oceans, but is apparently absent or rare (or undescribed) in the northern Indian Ocean, the tropical western Atlantic, the Great Barrier Reef region, and the Southern Ocean (Cornelius, 1975). The life cycle consists of three stages, in which the adult medusa releases either sperm or eggs forming relatively short-lived, lecithotrophic planula larvae. The planulae settle on to a substrate and metamorphose into hydroids. Hydroids are colonial, and asexually produce medusae, which are released into the plankton, completing the cycle. Additionally, 0. geniculata hydroids can reproduce asexually by releasing propagules or "capsules" of tissue (Bilard, 1904; Berril, 1948; Panteleeva, 1999). 0. geniculata hydroids are found from the intertidal down to about 100 m depth, growing on a variety of substrates especially brown algae and occasionally other invertebrates and fish (Cornelius, 1995). This species has the potential for extensive dispersal through planktonic medusae and planulae, or through hydroids rafting on drifting seaweeds (Cornelius, 1992). The goals of this research were to 1) examine genetic variation and phylogeography in Obelia geniculata using portions ofthe mitochondriall6S rDNA and COI genes; 2) determine the substitution rates of L6S rDNA and COI by calibrating nucleotide substitutions to the opening of the Bering Strait, and compare these rates with those in other invertebrates; and 3) investigate phylogenetic patterns and divergence relative to the last glacial maximum in the North Atlantic.

Materials and methods Sample collection and DNA sequencing Hydroids of Obelia geniculata (Linnaeus, 1758) were collected or obtained from colleagues from the North Atlantic and Pacific (Table 1) and preserved in 95% ethanoL. Hydroids were identified by A. Govindarajan or P. Schuchert, and vouchers for most samples are available upon request to A. Govindarajan. Genomic DNA was extracted using the DNEasy kit (Qiagen). Portions ofthe mitochondrial L6S rDNA and cytochrome

119 c oxidase subunit I (COI) were amplified under standard PCR conditions using the primers of Cunningham and Buss (1993) and Folmer et aI., (1994) (LC01490 and HC02198), respectively. PCR products were visualized on an agarose gel with ethidium bromide and purified with PCR purification kits (Qiagen). Purified products were cycle- sequenced with either Big Dye 2 or 3 sequencing chemistry (ABI) following the manufacturer's protocol, purified on a Sephadex column, and sequenced in both directions on an ABI 377. Sequences were aligned using Clustal X (Thompson et aI., 1994) and confirmed by eye with MacClade (Maddison and Maddison, 2000), although there was virtally no length variation in the sequences (only one indel). ModelTest (Posada and Crandall, 1998) was used to determine the best-fit model for maximum likelihood analyses (described below) conducted with P AUP* 4.0b 1 0 (Swofford 2000). For calculations of substitution rates and divergence estimates, model parameters under the best-fit model were estimated using maximum likelihood in PAUP*.

Phylogeny Because the mitochondrial genome represents a single locus, the 16S rDNA and COI sequences were combined for each individual sequenced and considered a single mitochondral haplotype. A maximum likelihood phylogeny was generated from a heuristic search with TBR branch swapping using the ModelTest parameters (corresponding to an HKY85 + I + G model). The starting tree was obtained from the set of most parsimonious trees found from a heuristic search (starting trees obtained via stepwise addition using 10 random addition replicates). Support for the nodes was estimated by conducting a likelihood bootstrap analysis with 300 replicates (identical haplotypes excluded to save computational time).

Substitution rates

Substitution rates were calibrated using P AUP* 4.0b 1 0 (Swofford, 2000) by calculating the average length of the central internal branch of a likelihood phylogeny using the best-fit model between the Pacific and the North Atlantic (New Brunswick,

120 Icelandic, and French) populations (to get gene, rather than population, divergence, Edwards and Beerli, 2000). A likelihood ratio test (Huelsenbeck and Rannala, 1997) was

used to see if the hydroids were evolving in a clock-like fashion. Likelihood scores were calculated in P AUP* using the best-fit model with and without the molecular clock constraint, and were used to generate a X2 test statistic with n-2 degrees of freedom (n =

number of taxa). Midpoint rooting was used when the molecular clock was enforced.

For comparison, we similarly calculated the substitution rate for L6S rDNA in another hydrozoan, the genus Hydractinia (Hydrozoa, Anthomedusa, Hydractiniidae) using previously published sequences (Cunningham and Buss, 1993) and a dated biogeographic event (Cunningham et aI., 1991 ; Young et aI., 2002). This calculation had not been done previously.

Phylogeography and North Atlantic population ages Evidence for geographic subdivision was obtained by conducting an AMOV A between the three groups (North Atlantic, Japan, and New Zealand) and pairwise FST's . using Arlequin 2.001 (Schneider et aI., 2000). The presence of geographically-restricted groups ofhaplotyes can be used to estimate minimum population ages. This is of 'interest since several of our populations were in regions thought to have been covered by glaciers during the last glacial maximum. The age of a clade composed of an ancestral haplotype and its descendants can be

estimated using the methods of Sailard et aI. (2000). If these form a perfect star phylogeny, the age is easily estimated according to coalescent theory. The more these haplotypes depart from a star phylogeny, the wider the confidence limits. First, the ancestral haplotype was identified by the method of Castelloe and Templeton (1994) which estimates outgroup weights based on haplotype frequency and connectivity. The haplotype with the highest outgroup weight is most likely the oldest. Following Sailard et aI. (2000), the divergence estimate p is the average number of links in terms of observed substitutions between the observed haplotypes from the ancestral haplotye:

121 p = (nili + n2h+ + nmlm)/n) where n = number of individuals with a given haplotype, 1 = number of steps (links) of a given haplotype to the ancestral haplotype, and m = number ofhaplotypes. The variance a is described by:

0'2 = (ni2ii + nlh + +nm2lm)/n2

The star index p/na2 (Torroni et aI., 1998; Sailard et aI., 2000), where a value of one equals a perfectly starlike phylogeny, was calculated. As described above, the more starlike, the smaller the confidence intervals. Since p is expressed in terms of number of observed substitutions, a per-locus rate of substitution is necessary. This is obtained by multiplying the substitution rate times the number of positions in the combined L6S rDNA and COI data.

Results Sample collection and DNA sequencing A total of 5 lObelia geniculata hydroids were collected and sequenced from New Zealand, Japan, and four locations in the North Atlantic (Massachusetts, New Bruswick, Iceland, and France). Four hundred fort base pairs of L6S rDNA and 575 base pairs of COI were sequenced for all taxa (GenBank accession numbers A Y530328-A Y530429). The dataset consisted of 1015 characters, 941 which were constant, 21 which were variable but parsimony-uninformative, and 52 which were parsimony-informative. The alignments were deposited in the EMBL-Align database (accession numbers ALIGN_000710 and ALIGN_00071 1 for 16S and COI, respectively).

Phylogeny

122 A total of 31 mitochondrial haplotypes were found. All Japanese individuals, and all but one New Zealand individual, were unique. The likelihood topology indicated three strongly supported (96-100%), reciprocally-monophyletic clades separated by long branches: one for the North Atlantic, one for Japan, and one for New Zealand (Figure 1). The arrangement of the three clades with respect to each other is unresolved. L6S rDNA and COI substitution rates. Minimum substitution rates were calculated by comparing divergence between North Atlantic and Japanese populations. Three ages for the trans-Arctic interchange were used: the minimum and maximum estimate for the initial opening of the Bering

Strait (3.1 and 4.1 mya) (Marincovich and Gladenkov, 2001) and 3.5 my a (Vermeij, 1991). Because these dates represent the initial opening, the resulting substitution rate estimate is the minimum possible rate. To obtain divergence estimates, a maximum likelihood phylogeny was constructed using the best-fit modeL. The length of the internal branch between populations estimates the actual age of divergence between populations, and serves to correct for polymorphism in the ancestral population (Edwards and Beerli, 2000). The best-fit models for each data partition (16S rDNA, L6S rDNA + COI, COI, COI 3rd codon posittons) and their estimated rates, are presented (Table 2). The molecular clock assumption was tested, but was not rejected (-In Lc10ck = 1946.40809, -In Lnoc1ock =

1913.82546, 2(-ln Lc10ck - -In Lnoc1ock) = 65.16526, d.f. = 49, critical value at 0.05 significance = 66.339). eomparison of mitochondrial substitution rates to other invertebrates The minimum substitution rates for 16S rDNA, COI, and COI 3rd codon positions were higher than those for anthozoans, and within the range of other marine invertebrates (Table 2, Table 3). The 16S rDNA rate of Obelia genicuZata was comparable to our estimated rate, based on published 16S rDNA sequences, of the hydrozoan Hydractinia spp. (Cunningham and Buss, 1993). The maximum date of divergence for Atlantic and Gulf of Mexico populations of the hermt crab host of

123 Hydractinia spp. is:: 4.1 millon years (Cunningham et aI., 1991; Young et aI., 2002). The divergence between Atlantic and Gulf of Mexico Hydractinia spp., using an HKY model is 0.01027, which yields a minimum substitution rate of 1.25 x 10-9 for Hydractinia 16S rDNA. This is lower than the Obelia geniculata rate, but both were considerably higher than published estimates for scleractinian corals (Romano and Palumbi, 1997; Table 3).

Phylogeography Within the North Atlantic, there appeared to be an ancestral haplotye. As expected (Castelloe and Templeton, 1994), the ancestral haplotype was deeply nested and was shared by Massachusetts (MA), New Brunswick (NB), and Iceland (IC) populations (Figure 2).

The AMOV A indicated that most (92.67%) of the variation corresponded to the three major clades found in the phylogeny (North Atlantic, JP, and NZ). 1.22% of the variation came from between localities within the three major groups (MA, NB, IC, FR,

JP, NZ), and 6.11 % of the variation came from within locations (Table 4). The pairwise FST values indicated significant, although in some cases small, genetic differentiation: only one pair (N-IC) was not statistically different (p ? 0.05, Table 5).

Estimates of population age Both the Icelandic and New Brunswick populations had a number ofhaplotypes not shared by other populations. In contrast, the Massachusetts sample was entirely composed ofhaplotypes shared with New Brunswick, consistent with a recent colonization from the north (Hewitt, 2000). If we consider the ancestral haplotype in the North Atlantic and its descendants that are restricted only to a particular population, we can estimate the minimum age of that population using the method of Sailard et ai. (2000). For New Brunswick the estimated age, 143 :: 47 kya, predates the last glacial maximum (20 kya). Including the

124 Massachusetts individuals does not change this result (150 :t 82 kya). The Icelandic population appears even older, at 204 :t 68 kya. To investigate the possibility that the trans-Arctic migration was more recent, thus moving the divergence dates to be more recent than the last glacial maximum, the effects of trans-Arctic migration date on divergence time from the ancestral haplotype and on the corresponding COI 3rd codon position substitution rate were modeled for the New

Bruswick samples (Figure 3). The more recent the date of trans-Arctic migration, the more recent the divergence from the ancestral haplotye and the higher the substitution rate. Trans-Arctic migration dates at or after ~ 750,000 years ago yielded divergence estimates at or after the last glacial maximum. However, the corresponding substitution rates were exceedingly high. For a trans-Arctic migration date of750,000 years ago, the COI 3rd codon substitution rate was 90.93 x 10-9 substitutions site-1 yeal-i, and increased rapidly as trans-Arctic migration dates became more recent.

Discussion We found three reciprocally-monophyletic clades of Obelia geniculata in New Zealand, Japan, and the North Atlantic. The New Zealand and Japanese populations, each collected from a single location, appear to have has much or more haplotype diversity as the four North Atlantic sampling localities combined. This is consistent with a more recent origin for the North Atlantic population, perhaps via an invasion from the Pacific during the trans-Arctic interchange that followed the opening of the Bering Strait 3.1 -4. 1 mya. However, additional data are necessary to determine the arrangement of the clades with respect to each other. Our substitution rate calibrations assume a trans-Arctic migration 3.1-4.1 mya, but do not depend on the direction of migration. An earlier opening 5.4 - 5.5 my a has been recently suggested (Marincovich and Gladenkov, 1999; Gladenkov et aI., 2002) but, unlike the later opening (Durham and MacNeil, 1967; Briggs, 1970; Vermeij, 1991), it does not appear to have been accompanied by a large faunal migration and so was not considered here. Many species, including molluscs, echinoderms, and algae are thought

125 to have been introduced to the North Atlantic from the North Pacific when the Bering

Strait opened up around 3.5 my a (Durham and MacNeil, 1967; Van den Hoek and

Breeman, 1990; Vermeij, 1991; Cunningham and Collins, 1998). Several species of the kelp Laminaria, a common substrate of Obelia geniculata, were among the invaders from the Pacific (Stam et aI., 1988). We used the opening of the Bering Strait to obtain a minimum estimate of substitution rate for Obelia geniculata. For both the COI and 16S rDNA genes, this rate is about an order of magnitude faster than rates estimated from the anthozoans (Table 3). The 16S rDNA rate for 0. geniculata is also higher than a rate estimated from published sequences for the hydrozoan genus Hydractinia (Table 3). The existence of three well-supported clades separated by long branches suggests that the North Atlantic, Japanese, and New Zealand populations have been separated for a long time. The three clades are reciprocally-monophyletic, suggesting they represent different species according to the phylogenetic species concept (A vise, 2000). Species distinctions in campanulariid hydroids, particularly in the genus Obelia, are controversiaL. Species-level taxonomy is based in a large part on theca (the chitinous covering) morphology, but environmental influences on morphology (e.g., Ralph, 1956) make identification difficult. In a revision of the genus Obelia, Cornelius (1975) synonomized about 120 species into 3. He later increased that number to 4 (Cornelius, 1990). However, unlike for other Obelia species, his treatment of 0. geniculata was not controversial

(Cornelius, 1990), and 0. geniculata is considered easily recognizable by the asymmetrical thickening of the perisarc, or exoskeleton (although the degree of thickening is variable). Nevertheless, our results suggest that diversity may be underestimated in 0. geniculata. Additional genetic studies wil be useful in resolving species boundaries in the genus Obelia. This study presents the first calibrated hydrozoan substitution rates. Because our rates are based on the maximum time of divergence, they represent minimum estimates. Obelia geniculata COI and L6S rDNA genes are evolving at a rate similar to other

126 invertebrates, countering the hypothesis that cnidarian mitochondria evolve slowly. Thus mitochondrial sequences should be a useful tool for studying hydrozoan phylogeography. Anthozoans exhibit very low substitution rates in their mitochondrial sequences, even at COI 3rd codon positions (which are under less selective pressure; Snell et aI., 1998; Medina et aI., 1999; France and Hoover, 2002). In Obelia geniculata, the divergence rate for third codon positions fell within the range for other invertebrates

(Wares and Cunningham, 2001).

Noting that sponges appear to have a slow rate of mitochondrial evolution, Shearer et ai. (2002) (citing R. Watkins pers. comm.) suggest that mitochondrial evolution was slow in ancestral metazoans and accelerated near the origin of the Bilateria. They point out that if a higher mitochondrial evolutionary rate were found in cnidarian classes other than the Anthozoa, then that accelerated rate likely evolved at the origin of the Medusazoa, as anthozoans are thought to be the basal cnidarians (Bridge et aI., 1992; Bridge et aI., 1995). Our results are consistent with this interpretation of a second independent origin of rapid mitochondral evolution. In addition to the trans-Arctic interchange, the Pleistocene glaciations are thought to have been a major force shaping the history of shallow-water flora and fauna in the North Atlantic by causing local extinctions, especially in the west (Ingolfsson, 1992; Cunningham and Collins, 1998). Our results suggest that it may have been important for

hydroids as welL. Although more comprehensive sampling is required, particularly in France, our divergence estimates which predate the last glacial maximum suggest that Icelandic and Canadian populations of Obelia geniculata may have survived in northern, glacial refugia. This conclusion is robust to the calibration of the molecular clock. In some cases, trans-Arctic invaders may have come more recently than after the initial opening of the

Bering Strait (Palumbi and Kessing, 1991; Van Oppen et aI., 1995). If this were the case for O. geniculata, the calculated Icelandic and New Brunswick divergence date would be overestimated because the substitution rate estimate would be too low. However, this seems highly unlikely because the minimum substitution rate necessary to push the

127 divergence dates to the last glacial maximum (requiring a maximum trans-Arctic migration date of ~750 kya) is considerably higher than the substitution rate in all other invertebrates compared except for Mytilus edulis (95.1 x 10-9 substitutions site-I year-I; Table 3). M edulis is known to have an exceptionally high mitochondrial evolutionary

rate, possibly due to its unusual form of mitochondrial ineritance (Hoeh et aI., 1996). Thus, it seems most likely that 0. geniculata had a trans-Arctic migration before 750,000 years ago, supporting our original estimate of pre-glacial ages for 0. geniculata populations in Iceland and the Canadian Maritimes.

The presence of unique haplotypes in 0. geniculata from Iceland and New Brunswick also add to the mounting genetic evidence for glacial refugia in these areas (Holder et aI., 1999; Dahlgren et aI., 2000; Wares et aI., 2002; Young et aI., 2002). Geological evidence suggests that unglaciated pockets may have existed in northern Norway and Scotland (Dawson, 1992, Siegert, 2001) and in the Canadian Maritimes and Georges Bank (Rogerson, 1983; Pielou, 1991, Holder et aI., 1999). These unglaciated pockets may have provided the refugia indicated by the genetic data. Our results also suggest a recent southward range expansion by two haplotypes from New Bruswick into Massachusetts (the Woods Hole, MA sampling site is located on the southern shore of the biogeographic boundary of Cape Cod (Franz and Merril, 1980 a, b D. As expected for a recent introduction, genetic diversity was lower than, and a subset of, the putative New Brunswick founding population (Hewitt, 2000). Similarly, Dahlgren et aI., (2000) suggested a southern range expansion for the bivalve Arctica islandica due to global climate change. This pattern is also seen in the gastropod

Littorina litorea, a species believed for a long time to have been introduced into the western Atlantic from Europe, but which is now thought to be a recent southward range expansion from Canada (Wares et aI., 2002). Such southward range expansions may be common in other invertebrate taxa as welL. Wares and Cunningham (200 l) suggest that species capable of long distance dispersal may have persisted through glacial periods because they could take advantage of widely scattered, ephemeral refugia (although the role of dispersal ability in

128 colonization is complex, Cuningham and Collins, 1998). The presence of a planktonic medusa stage in the life cycle of Obelia geniculata could provide a mechanism for substantial dispersaL Additionally, hydrozoans possessing a medusa stage may have relatively longer-lived planulae (Sommer, 1992). In 0. geniculata, several medusae generations are produced per year, and the medusae may live for several weeks (Kramp, 1927). Planulae of Obelia spp. may live from 5 d to 3 wk (Bodo and Bouilon, 1968). Dispersal can also occur via the attached hydroid stage by rafting on algae (Cornelius, 1992; Ingolfsson, 1995). Hydroids living on Laminaria spp. live as long as the blades to which they are attached, which may be up to 15 months (Kramp, 1927). Finally, dispersal may occur via asexual propagules (Billard, 1904; Berril, 1948; Panteleeva, 1999). Nevertheless, there was significant genetic variation despite the dispersal potential

of the planktonic medusae, planulae, propagules, and algal rafting. Not surpnsingly, because of the large geographic distances between them, there were large differences between the North Atlantic, Japanese, and New Zealand specimens indicative of cryptic

speciation. However, within the North Atlantic, the lack of variation in the Massachusetts

samples relative to the New Brunswick samples, and the presence of unique haplotypes in New Brunswick and Iceland, suggest that dispersal may also be restricted at relatively smaller scales. This is counterintuitive given the enormous dispersal potential of Obelia geniculata. Boero and Bouillon (1993) suggested that the presence of a medusa stage does not necessarily imply wide dispersal. In fact, they found that among Mediterranean hydrozoans, nominal species with apparently cosmopolitan distributions were less likely to have a medusa stage. Studies of other cnidarian medusae also suggest that a planktonic lifestyle does not lead to genetic homogeneity and that there can be considerable genetic differentiation and even cryptic speciation, as in the scyphozoan Aurelia aurita (Dawson and Jacobs, 2001). Oceanographic or behavioral barriers may be responsible for this restricted planktonic dispersal (Hamner et aI., 1994). Additional research is necessary to determine the actual, as opposed to potential, dispersal of the several species presently attrbuted to Obelia geniculata and its role in surviving the Pleistocene glaciation.

129 Acknowledgements We are grateful to Peter Schuchert, Lea-An Henr, Yayoi Hirano, and Bil Grossman for providing specimens, and to Ferdinando Boero, Larr Madin, Jesús Pineda, and Tim Shank for helpful comments. This work was supported by an NSF PEET grant to C. Cunningham (DEB- 9978131) and an Ocean Life Institute fellowship to K. M. Halanych. The experiments comply with the curent laws of the USA.

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137 Table 1. Obelia geniculata. Localities sampled for hydroids.

Locality Sample # Latitude Longitude Sample Date Code Samples

St. i\ndrevvs, ~evv NB 14 45005' 670 03' July 2002 Brunsvvick, Canada

Woods Hole, Mi\ 9 41032' -700 40' October 2001 Massachusetts, USi\

Roscoff, France FR 4 48° 43' _3° 59' i\pril 1998

Garour/Sandgerdi, IC 8 64° 04' - _22° 43' - May 2000 Iceland 64° 02' -22°39'

Misaki, Sagami Bay, JP 8 34° 19' 135° 09' September Japan 2002

Wellington, ~evv ~Z 8 -41 ° 18' 174° 47' December Zealand 2001

138 Table 2. Obelia geniculata. Range of divergence rates based on minimum (3.1 mya), maximum (4.1 mya), and commonly used (3.5 mya) estimates for opening of Bering Strait.

Gene Substitution rate Substitution rate Substitution rate Best-fit (substitutions sité yro!) (substitutions sité yro!) (substitutions sité yro!) model

(x 10-9) (x 1009) (x 1009) used in rate

3.1 my a 3.5 my a 4.1 my a calculation

16S 2.76 2.44 2.08 16S + 6.13 5.43 4.63 HKY + G cm cm 7.38 6.54 5.58 HKY + G cm 3rd 22.00 19.48 16.63 HKY codon

139 Table 3. Obelia geniculata. Substitution rates of 16S rDNA and COI (all positions) compared with those from other cnidarians (0. geniculata in bold). Substitution rate for cytochrome b from an anthozoan is included for comparison, although this gene was not sequenced in 0. geniculata. 0. geniculata COI 3rd codon position substitution rate is compared with other invertebrates calculated by Wares and Cunningham (2001) with the F84 modeL. Even ifthe unlikely earlier, 5.5 my a opening (which was not accompanied by a large faunal migration) is considered, 0. geniculata rates would be lower, but still higher than anthozoans (16S rDNA: 1.55 x 10-9 substitutions site-! yr-!; COI: 4.16 x 10-9 substitutions site-! yr-!; COI 3rd codon positions: 12.40 x 10-9 substitutions site-! yr'!).

Gene Phylum Species Substitution Calibration Source rate reference (sub sité yr-1)

(x 10-9) 16S Cnidaria O. geniclilata 2.44 Bering Strait Present study (Hydrozoa) (3.5 mya) 16S Cnidaria Hydractinia 1.25 Florida (4.1 Cunningham et aI., (Hydrozoa) mya) 1991; Young et aI., 2002 16S Cnidaria Scleractinian 0.1-0.6 Fossil record Romano and (Anthozoa) corals Palumbi 1997

COI Cnidaria O. geniclilata 6.54 Bering Strait Present study (Hydrozoa) (3.5 mya) cm Cnidaria Montastraea 0.5 Fossil record Medina et aI., 1999 (Anthozoa) spp. Cytb Cnidaria Acropora spp. 0.5-0.9 Fossil record Van Oppen et aI., (Anthozoa) 1999 cm 3rd Cnidaria O. geniclilata 19.5 Bering Strait Present study codon (Hydrozoa) (3.5 mya) cm 3rd Arthropoda Alpheus spp. 19 Isthmus of Knowlton and codon (Malacostraca) Panama Weigt 1998 cm 3rd Arthropoda Sesarma spp. 21 Isthmus of Schubart et aI., codon (Malacostraca) Panama 1998 COI 3rd Mollusca Littorina 24 Bering Strait Wares and codon (Gastropoda) obtusata (3.5 mya) Cunningham 200 1 cm 3rd Arthropoda Semibalanus 27.6 Bering Strait Wares and codon (Maxilopoda) balanoides (3.5 mya) Cunningham 200 1 cm 3rd Arthropoda Idotea 36 Bering Strait Wares and codon (Malacostraca) balthica (3.5 mya) Cunningham 2001 cm 3rd Arthropoda Euraphia spp. 38 Isthmus of Wares 2001 codon (Maxilopoda) Panama cm 3rd Mollusca Nucella 44.3 Bering Strait Wares and codon (Gastropoda) lapilus (3.5 mya) Cunningham 2001 cm 3rd Echinoderma Asterias 48.4 Bering Strait Wares and codon (Asteroidea) rubens (3.5 mya) Cunningham 2001 cm 3rd Mollusca Mytilus edulis 95.1 Bering Strait Wares and codon (Bivalvia) (3.5 mya) Cunningham 2001

140 Table 4. Obelia geniculata. AMOV A results. Groups represent the three major clades: North Atlantic, Japan, and New Zealand. Populations represent the 6 populations: Massachusetts (MA), New Brunswick (NB), Iceland (IC), France (FR), Japan (JP), and New Zealand (NZ).

Source or variation d.r. Sum or squares Variance components Percentage or variation Among groups 2 408.972 16.47575 92.67 Among populations 3 8.650 0.21734 1.22 within groups Within populations 45 48.907 1.08682 6.11 Total 50 466.529 17.77990

141 Table 5. Obelia geniculata. Pairwise FsT's and associated p-values (in parentheses).

MA NB IC FR JP NB (0.00000) IC 0.42176 0.03547 (0.00000) (0.13514) FR 0.49671 0.19653 0.14118 (0.00901) (0.01802) (0.03604) JP 0.95258 0.94038 0.92409 0.89158 (0.00000) (0.0000) (0.00000) (0.00901) NZ 0.95557 0.94050 0.92468 0.89326 0.89655 (0.00000) (0.0000) (0.00000) (0.00000) (0.00000)

142 JP 96 JP JP JP JP JP JP JP

97 NZ NZ

NZ

FR-4 100 NB-13NB-14 FR-3 NB-12 NB-1 NB-2 NB-3 NB-4 NB-5 NB-6 NB-7 NB-8 MA-1 IC-1 IC-2 IC-3 IC-4 IC-5 IC-6 IC-7 IC-8 FR-2 FR-1 NB-9 NB-10 NB-11 MA-2 MA-3 MA-4 MA-5 MA-6 MA-7 MA-8 MA-9 - 0.001 substitutions/site

Figure. 1 Obelia geniculata. Maximum likelihood topology. Numbers indicate parsimony bootstrap values (300 replicates). Only bootstrap values for the three major clades are shown. Five nodes within these clades had bootstrap values of 50 to 71, and the rest were 0: 50. North Atlantic sample names correspond to sample names in Figue 2.

143 144 ~L

E5 8

Figure 2. Obelia geniculata. Parsimony network for North Atlantic samples. A heuristic search yielded 6 most parsimonious trees, all with 23 steps; the tree identical to the likelihood topology (Figure 1) is represented here. The ancestral haplotye is shaded. Sample names correspond to Figue 1 Sample NB-1l is separated from the NB-MA haplotye by one step; because that change was a deletion, it was coded as missing data in the likelihood analyses and not indicated on Figure 1

145 146 120000 800

700 100000 __Divergence (lower bound) -- COI codon I ~sition substitution rat 600 :;c: '" II e 80000 'C c. '" e 500 '" ~ ,g Ë¡ '" c ëñ- .Q 0-'" I! 60000 400:ø ~o "' .E 0 T' ..'" '" ~~~ '- ::- c: 300 en ~ Ql ~ 40000 - .. Ql ~ i5 200

20000 100

0 o co; co; co; co; co; co; co; co; co; co; CI: CI: CI: ,,1:cl: ",1:cl: cç§ ,,1:cl: ~1:cl: cl: cl: ai " ",\1: 'i.s 'i~ 'i ";r;1: ";C\1: Trans-Arctic migration (years before present)

Figure 3. Obelia geniculata. Effect of trans-Arctic migration time on divergence of the New Brunswick samples (which diverged more recently than the Icelandic samples) from the ancestral haplotype and on the substitution rate of COI 3rd codon positions (which can be compared to a variety of other invertebrates). To be conservative, the lower bound (mean minus one SD) ofthe divergence estimate was used. A: assuming migration at the initial opening, 3.5 mya, results in divergence well before the last glacial maximum and a substitution rate as described in the text and Table 3; B: assuming migration at or after 750,000 years ago results in divergence approximately at or after the last glacial maximum (20,552 years ago) and an exceptionally high substitution rate (90.93 x 10-9 substitutions site"i year"i).

147 148 Chapter 5 Conclusions

Summary This thesis has investigated aspects of the evolution, systematics, and phylogeography of the Campanulariidae, which are an important, abundant, and widely distributed group of hydrozoans with a variety of developmental modes and life history strategies. An introduction to the Campanulariidae was provided in Chapter 1, which reviewed their life cycles, development and taxonomy. The important issues in campanulariid biology were outlned, and include: the frequency oflife cycle transitions and their ecological and taxonomic consequences, the possibility of an evolutionary reversal and re-invention of the medusa stage, and species-level taxonomy. The specific aims of this thesis were to: 1) investigate the frequency oflife cycle transitions and the possibility that Obelia medusae are re-invented by constrcting a molecular phylogeny of the Campanulariidae and reconstructing ancestral character states; 2) examine the classification of the Campanulariidae II light of the molecular phylogeny at the family, genus, and species levels, and make the appropriate taxonomic revisions; and 3) examine the phylogeography and mitochondrial evolution in the campanulariid Obelia geniculata. In Chapter 2, a molecular phylogeny was constructed using 2 nuclear (L8S rDNA and calmodulin) and 2 mitochondrial (16S rDNA and cytochrome c oxidase I (COlD genes. The genes were analyzed together and separately, and most of the phylogenetic signal came from the L8S rDNA. The calmodulin, L6S rDNA, and COI trees were very poorly resolved by bootstrap methods. The Campanulariinae and elytia lineages appeared monophyletic, and there were at least 2 obeliinid clades. The placement of Obelia bidentata was problematic, probably due to its very long branches. The arrangement of the major clades with respect to each other was not resolved. Life cycle tyes were traced on to the combined phylogeny and the ancestral states reconstrcted using parsimony. A sensitivity analysis was conducted to examine the effects of weighting gains greater than losses, and found that even when only a small gain cost was

149 applied, the fixed gonophore-bearing Laomedea likely derived multiple times from an ancestor with Obelia medusae, rather than vice versa, and Obelia may have derived from an ancestor with tyical medusae. The results depended on the underlying phylogeny, however. The taxonomic results of the combined molecular phylogeny presented in Chapter 2 were discussed in Chapter 3. Some aspects of the traditional taxonomy were supported, while many others were refuted. Bilardia, referred to the Campanulariidae by some, fell well outside the campanulariid clade. Several species of Bonneviella (Bonneviellidae), originally designated as outgroups, were deeply nested within the otherwise monophyletic Campanulariinae lineage. Diagnoses of the Campanulariidae and

Campanulariinae were modified accordingly. The taxonomic approach of using life cycle type to define genera was not supported in the Obeliinae, and the obeliinid genera were merged into a single genus, Obelia. At the species level, some nominal cosmopolitan species were obtained from several locations, and of these, elytia gracils appeared to represent multiple cryptic species, while Obelia longissima may be truly cosmopolitan. Finally, the putative meconidia (medusoids) of Gonothyraea (Obelia) inornata were shown to be simple acrocysts (external capsules) without any medusoid features. Chapter 4 examined the phylogeography and mitochondrial evolution of the widely distributed campanulariid Obelia geniculata. Phylogeographic studies commonly use mitochondrial DNA markers; however, in cnidarians (mainly anthozoans), the substitution rate is exceptionally low, limiting its utility. Sequence divergence of the mitochondrial 16S rDNA and COI genes in Obelia geniculata from the North Atlantic and North Pacific was calibrated against the opening of the Bering Strait, approximately 3.5 million years ago, resulting in the first calibrated substitution rates for a hydrozoan. Substitution rates were considerably faster than in anthozoans, and comparable to other invertebrates. Analysis of Obelia genicuZata 16S rDNA and COI from New Zealand, Japan, and the North Atlantic revealed that the three localities were reciprocally monophyletic and separated by long branches, consistent with what would be expected for crytic species. Within the North Atlantic, divergence ofIcelandic and New

150 Bruswick haplotyes from the ancestral haplotype appeared to occur well before the last glacial maximum, suggesting that populations there did not go extinct during the last glaciation, but may have existed in refugia. Hydroids from Woods Hole, MA, USA appeared to be a relatively recent introduction from the north. The appendix includes a related side project on species identification in the endosymbiotic bivalve-inhabiting hydro ids of Eugymnanthea (Hydrozoa, Leptomedusa,

Eirenidae). These hydro ids occupy an unusual habitat, inside bivalves of many species, particularly Mytilus galloprovincialis. They are found only in the Mediterranean and Japan, where they have been discovered only relatively recently. Their disjunct distribution, morphological similarity, and the observation that Mytilus galloprovincialis was likely introduced to Japan from the Mediterranean have lead to continuing doubt over whether the two localities represent different species, or if the Japanese hydroids were introduced to Japan from the Mediterranean with their hosts. Reciprocal breeding crosses showed that Italian and Japanese Eugymnanthea could not interbreed, indicating they are different species according to the biological species concept. Analysis of their L6S rDNA sequences showed that the two forms were reciprocally monophyletic, and diverged by about 12%. A tissue grafting test, previously proposed to identify species, could not distinguish Italian and Japanese specimens. Finally, while hydroids from Italy and Japan were morphologically indistinguishable, there were usually slight differences in the medusoid stage. The results confirm that the two forms are different species, and that the Japanese form may not have resulted from an introduction from Italy.

Broader impacts and future directions This thesis provided new insight into many aspects of hydrozoan evolution and systematics, and has helped resolve some controversies within the Campanulariidae while suggesting new avenues for futue research. Chapter 2 presented the first molecular phylogeny of the Campanulariidae, demonstrating the utility of genetic markers that are relatively independent of the life cycle and environmental biases that plague hydrozoan morphological analyses. Future studies with additional taxa and genetic markers may

151 help to determine the arrangement of the major campanulariid lineages which were not resolved here. The phylogeny indicated multiple life cycle transitions within the Obeliinae, suggesting that they are more frequent than sometimes assumed. Studies of other marine invertebrates with complex life cycles suggest several ecological correlates

(e.g., dispersal, temperatue, latitude, size) to the presence and duration of a planktonic stage, and these should be further examined in the Campanulariidae and other hydrozoans. It seems likely that the ancestral Obelia may have possessed a typical medusa rather than medusoids or fixed gonophores, although alternatives cannot be completely ruled out. More research is necessary to better resolve the underlying phylogeny, and to understand the genetic and developmental changes involved in life cycle transitions. If an ancestor with typical medusae is supported in future studies, it will be interesting to determine the mechanism(s) responsible for effecting so many morphological and developmental changes. For example, similarities between some Obelia medusa features to hydranth features indicate that hydranth developmental pathways are involved in Obelia medusa development. Gene expression studies may be particularly helpful in understanding the genetic changes and pathways involved, the relative ease of gains versus losses, and the likelihood of evolutionary reversals. The molecular phylogeny presented in Chapters 2 and 3 indicate the Campanulariidae and indeed, the entire Leptomedusae, may need revision. The exclusion of Bilardia and especially the inclusion of the Bonneviellidae demonstrated how genetic markers can reveal unexpected relationships. Results also showed that the taxonomic approach of using life cycle type as a generic character can lead to paraphyly and that DNA sequences can be helpful in delineating species boundaries. Traditional morphological taxonomy is exceptionally difficult in the Hydrozoa, largely due to their complex life cycles and phenotypic plasticity. In fact, a unified classification for hydro ids and medusae was not even proposed until 1960, and difficulties persist, with several examples of hydro ids with features characteristic of one family and their corresponding medusae characteristic of another. Molecular studies, like the one conducted in this

152 thesis, wil be useful in resolving controversies in hydrozoan classification at many levels. Results from several chapters and the appendix suggest that species-level biodiversity may be underestimated in the Campanulariidae and other hydrozoans, although some true cosmopolites may exist. This is in contrast to the most recent monographs on the Campanulariidae, which synonymized numerous nominal taxa. As in other invertebrates, genetic studies wil be crucial in identifying crytic species. When possible, breeding experiments can corroborate results from DNA studies, and careful morphological examination may identify distinguishing featues. Finally, the first calibrated hydrozoan mitochondrial substitution rates presented in Chapter 4 revealed that hydrozoan mitochondria may be evolving considerably faster than anthozoans, at a rate comparable to other invertebrates. This indicates that mitochondrial markers wil be useful in phylogeographic studies ofhydrozoans, as they are in many other invertebrates. Analyses of Obelia geniculata L6S rDNA and COI revealed likely cryptic speciation in Japan, New Zealand, and North Atlantic. The relationship between these three clades was not resolved, however, so future studies incorporating samples throughout the entire nominal 0. geniculata range wil be helpful in reconstructing the history of this group. Furthermore, the data added to a growing body of evidence that refugia in the North Atlantic existed during the last glaciation, where species that may have went extinct elsewhere could have survived. Similar studies on other hydroids with trans-Atlantic distributions can help determine the extent of this phenomenon.

153 154 Appendix

Species identification of bivalve-inhabiting marine hydrozoans of the genus Eugymnanthea*

*Manuscript based on this appendix coauthored with Cinzia Gravili, Stefano Piraino, and Shin Kubota has been accepted for publication in Invertebrate Biology.

Abstract Species-level identification is difficult in the symbiotic bivalve-inhabiting hydrozoans of the genus Eugymnanthea (Cnidaria, Hydrozoa). Morphological differences are detected only in the adult medusoid stage. Eugymnanthea is known only from the Mediterranean and the western Pacific, and doubt persists over whether the two localities are inhabited by different species. Because the bivalve host, Mytilus galloprovincialis, is thought to be introduced by humans from the Mediterranean to the western Pacific, there has been speculation that the Mediterranean Eugymnanthea was also introduced along with its host. Here, we evaluate the species status of the two forms with breeding experiments, morphology, and two recently developed tools for discrimination. a mesoglea cell adhesion and spreading test, and L6S rDNA comparison. Reciprocal crosses of the two forms failed to produce normal offspring, providing evidence that they are indeed different species according to the biological species concept, and suggesting that the Pacific form is not an invasion of the Mediterranean form. The tissue-grafting test failed to distinguish between the two forms, while the morphological and genetic evidence corroborated the breeding results.

Introduction Many marine hydroids of the family Eirenidae (Hydrozoa, Leptomedusae) occupy an unusual habitat, living in the mantle cavity of some bivalve molluscs, including several species used for human consumption: Mytilus galloprovincialis (Cerrti, 1941;

Crowell, 1957; Kubota, 1992a), erassostrea virginica (Kubota and Larson, 1990), and

TiveZa mactroides (Narchi and Hebling, 1975). Details ofhydroid-bivalve associations

155 are reviewed in Kubota (1983), Kubota (1987a); and Piraino et ai. (1994). In some areas, the frequency of symbiotic hydroids can be quite high; for example, in Taranto, Italy, up to 86% of M galloprovincialis contained hydro ids (Kubota, 1989). However, the percentage of infected mussels can vary considerably with location, mussel size, and season (Kubota, 1983; Piraino et aI., 1994). M galloprovincialis cultivation is an important industr in Taranto, and the high incidence of infection by the eirinid Eugymnanthea inquilina there has led some local fisherman to blame mussel death on the hydroids (although this mortality was ultimately blamed on a dinoflagellate bloom combined with high temperature and oxygen depletion). Despite this, Piraino et ai. (1994) found that this hydroid-bivalve relationship is probably mutualistic. The hydroids receive habitat, protection from predators, and a food supply. The mussels may receive some protection against trematode parasites as the hydroids can ingest their sporocysts, and trematode infection is very low in mussels with hydroids (Piraino et aI., 1994). Additionally, Tiscar (1992) found no effect ofhydroid attachment on mantle tissue. Members of the eirenid genus Eugymnanthea have a complex life cycle: the adult (sexual) stage is a motile medusoid, with some, but not all, characteristics of fully functional hydrozoan medusae (Naumov, 1960). The medusoids can be gonochoric or rarely hermaphroditic (Celiberti et aI., 1998; Kubota in press), and fertilization and early developmental stages may still occur within the subumbrellar cavity (Celiberti et ai. 1998). The planula larvae settle in a host bivalve, and develop into hydroids, which complete the cycle by budding medusoids (Figure 1). Hydroids can attach to the mantle, labial palp, and foot of the host bivalves. In Japan, hydroids can also attach to the gill, although this is rarely observed in Italy (Kubota, 1989). Instead of a stolon as in other hydroid species, Eugymnanthea have a sucker-like, non-penetrating pedal disk that is used for attachment (Kubota, 1983). Species-level identification of Eugymnanthea is problematic unless the matue medusoids can be cultured. In the past, it was thought that there was only a single species; however, Kubota (1991a) raised the medusoids of Japanese and Italian specimens, and based on differences in the manubrium and statoliths, he considered them

156 two distinct species (E.japonica andE. inquilna). Kubota (1989) was unable to successfully interbreed the two forms; however, his mutual crossing was only in one direction. In his experiments, he crossed four Mediterranean males with five Japanese females. Only one of these crosses produced offspring, and those degenerated after the third day of the experiment. To date, Eugymnanthea has only been discovered in the Mediterranean Sea and the western Pacific Ocean (Kubota, 2000). E. japonica is found in Japan and Taiwan, inhabiting primarily the bivalves Mytilus galloprovincialis, erassostrea gigas, Barbatia virescens, and occasionally other species (Kubota, 1992a; Kubota, 2003). E. inquilna is found along the northern coast of the Mediterranean Sea (Italy, France, Spain, Croatia, Greece), and, like E. japonica, is found primarily in the mussel M. galloprovincialis and occasionally in other species (Piraino et aI., 1994; Kubota, 2000; Rayyan et aI., 2002; Kubota, in press). The disjunct geographical distribution and the overlap in bivalve host species has resulted in lingering doubt about the species status of the "two forms" and continuing speculation that the Japanese form may have been introduced from the Mediterranean via transfer by the host (Kubota, 1979). It is thought that M. galloprovincialis was transported to Japan, initially to the region of Kobe between 1930- 1935 (Wilkens et aI., 1983; Koehn, 1991, Kubota et aI., 1995), possibly from the Mediterranean (Wilkens et aI., 1983) and the hydroids could have been introduced along with their hosts. E. inquilna, first recorded by Palombi (1935), has a relatively long history in the Mediterranean, but E.japonica was only discovered in Japan in 1979 (Kubota 1979), despite a relatively long history of research on bivalve-inhabiting hydroids in that country. Furthermore, Its range in Japan has been expanding. Originally it was found in only one location (Shimoda, central Japan), but was subsequently discovered in many other locations in southern Japan (Kubota, 1991a; Kubota, 1 992a) and Taiwan (Kubota et aI., 1999). Explanations for this range shift include: 1) the hydroids are native to the Mediterranean and were introduced to Japan with their host M. galloprovincialis, and have subsequently spread from the site(s) of introduction; 2) the hydro ids are native to

157 Japan but were previously rare, and the introduction of M galloprovincialis provided a new host and facilitated their spread; and 3) the hydro ids are native to Japan but were previously rare, and other factors, such as habitat modification, have improved environmental conditions and facilitated their spread (Kubota, 1992a). In order to determine the origin of Eugymnanthea japonica, it is necessary to resolve its species-level taxonomy. Various methods have been proposed for species- level identification of cryptic species (Knowlton, 2000), and DNA sequences have proven useful for many taxa. For hydrozoans, the L6S rDNA may be useful to distinguish species (Cunningham and Buss, 1993; Schierwater and Ender, 2000; Govindarajan et aI., submitted). Alternatively, Schmid et al. (1992) demonstrated that the mesoglea test, which is based on the adhesion and spread of cells on the mesoglea, may be an effective tool with which to discriminate among hydrozoan species. In this test, tissue fragments from one individual are grafted onto mesoglea from another individuaL. The tissue fragments should only adhere and spread if the mesoglea is from a closely related (i.e., conspecific) individuaL. The goal of this research was to resolve the controversy surrounding Eugymnanthea. Morphological and molecular evidence was used 1) to confirm that the two forms are indeed separate species; 2) to assess two methods (the mesoglea test and

L6S rDNA sequences) of identifying species accurately and through all life cycle stages; and 3) to deduce whether or not the Japanese form was introduced from the Mediterranean Sea.

Methods eollection, culture, and morphological observations

E. inquilna was obtained by collecting mussels (M. galloprovin cia lis ) from two localities along the southern coast ofItaly: Lago Fusaro (40049'N, 14°03'E; the tye locality) and Taranto (40028'N, 17°l4E). The mussels were brought back to the laboratory, where some of them were opened and the hydroids, if present, carefully removed. The hydro ids were cultured, with or without their hosts, in small trays or dishes

158 filled with natural seawater (salinity = 38--40 %0). These rearing vessels were kept under constant conditions of23 °C and 15L:9D photoperiod, and the animals were fed once daily with newly hatched brine shrimp nauplii. The water was changed daily and was continuously aerated. When medusoids were released from the hydroids, all were collected, cultured, and examined daily with a microscope. For all of the medusoids, the primary diagnostic features ( the number of statoliths per statocyst and the presence or absence of a manubrium) were recorded. Rarely, when immediate observation was not possible, the newly liberated medusoids were preserved in buffered 10% formalin and examined later. Some hydroids were preserved in 99% ethanol for subsequent DNA analysis. In Japan, E. japonica was collected from the mussel (M galloprovincialis) attached to rafts moored in two localities in Japan facing the Pacific Ocean: Atami, Shizuoka Prefecture (35°05'N, 139°05'E; very close to the type locality in Shimoda) and Shirahama, Wakayama Prefecture (33°42'N, 135°22'E). The hydroids (and subsequent medusoids) were collected, cultured, and analyzed in the same manner as for E. inquilina.

Additional observations from Kubota (1989; 1991a) were also used for comparison.

Breeding eXperiments For each crossing, one pair, i.e. one male and one female, was prepared. Mature medusae of male E. inquilina and female E. japonica, and female E. inquilina and male E. japonica, were placed in culture dishes for spawning. Each pair was in a separate culture dish. Pairs of conspecific male and female medusae were also placed together for a control. Three conspecific and 13 interspecific crosses were conducted. Spawmng was triggered by light exposure, as male and female medusae of both species release their gametes after about half an hour after the light is turned on (Kubota, unpublished data). Hermaphroditic medusae, easily distinguished by the presence of mature sperm and eggs within the same gonad, were not used, as they may self-fertilize (Celiberti et aI., 1998).

The presence of late-stage planula larvae (with differentiated endoderm and nematocysts) (Kubota, 1991b) indicated successful mating.

159 Mesoglea test The mesoglea test was performed using live specimens. In this cell adhesion and spreading test on isolated ECM (extracellular matrix, or mesoglea) was carried out following the methods described by Schmid et al. (1992) and Reber-Müller et al. (1994). To summarize, the ECM was excised and the cells were allowed to dissociate from it by incubating in a Ca2+Mg2+ --free seawater. Cell dissociation was facilitated by blowing on the ECM with a pipet. The ECM was then washed in distiled water, placed on a coverslip, and allowed to dry. Tissue from the test sample was then excised, washed in filtered seawater, and applied to the ECM. After about an hour, the ECM was examined for cell adhesion. Tissue of Eugymnanthea was also grafted on to the ECM of Podocoryna carnea (Hydrozoa, Anthomedusae), a phylogenetical1y distant hydroid species (collected from Naples, Italy and maintained in the Schmid laboratory) as a control.

DNA sequencing

A ~600 bp portion of the L6S rDNA gene was sequenced. DNA was extracted from ethanol-preserved tissue belonging to 3 E. japonica and 4 E. inquilna with the DNEasy Kit (Qiagen) according to the manufacturer's protocol and amplified with hydrozoan-specific primers (Cunningham and Buss, 1993) following standard protocols

(Palumbi, 1996). The PCR product was visualized on a 1 % agarose gel with ethidium bromide, and was purified with the PCR purification kit (Qiagen). The purified product was cycle-sequenced using Big Dye 2 sequencing chemistry (ABI) following the manufacturer's protocol. The cycle-sequenced product was purified on a Sephadex column and sequenced on an ABI 377 Sequencer. DNA sequences were aligned using ClustalX 1.8 (Thompson et aI., 1997), and the alignment was confirmed by eye. Sequences were analyzed with MacClade 4.0 (Maddison and Maddison, 2000) and

P AUP* 4.0b 1 0 (Swofford, 2002) using equally-weighted parsimony (details in figue

160 legend). Unique sequences were submitted to GenBank (accession numbers AY285162-- AY285164).

Results Morphological observations The number of statoliths per statocyst was counted in 1555 medusoids of E. inquilina from Taranto, Italy and compared with observations on 4597 medusoids of E. japonica from Kubota (199la) (Table 1a). Each medusoid of E. inquilna typically had 8 statocysts (2 per interradial sector), but occasionally there were variable numbers of radial canals (3,5, or even 6) and statocysts (5--12) Some statocysts from formalin- preserved medusoids had damaged statoliths, and these were excluded from our analysis. Statolith numbers among statocysts of the same specimen could vary by 1, or rarely 2 or more, statoliths. A total of900l undamaged statoliths were examined in E. inquilna. The number of statoliths/statocyst ranged from 0--8, and the mean was 2.4 :10.83. In this species, fifty - four percent of the statocysts had 1 --2 statoliths, which is the statolith number found in 97% of statocysts of E. japonica (see tables la, lb). More generally, the number of statoliths per statocyst was significantly different from E. japonica (Kolmogorov- Smirnov test, p ~ 0.01), and only 33 medusoids (2.1 %) had both l--2 statoliths per statocyst and a manubrium.

Breeding experiments

All conspecific mating crosses produced healthy, normal planulae, but none of the 13 interspecific crosses did (Table 2). However, some of the interspecific crosses resulted in a few abnormal, short-lived, rotating or swimming larvae without nematocysts and with or without endoderm. These may have been partenogenetically produced (Kubota, 1987b; Kubota, 1987c; Kubota, 1991b) and all died within a few days.

161 Mesoglea test The ectodermal tissue grafted from medusa bud of E. japonica adhered to the mesoglea of both other E.japonica andE. inquilna, but not to the mesoglea of the distantly related Podocoryna carnea (Table 3). Ectoderm grafted from E. inquilna adhered to the mesoglea of other E. inquilna, but not to P. carnea. However, E. inquilna tissue was not grafted on to mesoglea of E. japonica, because, at this point, it was apparent from the previous test (graft of E. japonica on to mesoglea of E. inquilna) that this method could not distinguish between the two.

DNA sequencing Five hundred seventy-one base pairs were sequenced and aligned for three individuals of E. japonica (two from Atami and one from Shirahama) and four individuals of E. inquilna (3 from Taranto and 1 from Lago Fusaro). Of these, 78 characters were variable, including 70 parsimony-informative sites. The three sequences of E. japonica were identical, and two (including the Lago Fusaro individual) out of the four sequences of E. inquilna were identicaL. A third differed by one base pair, and the fourth differed from the first two by 14 base pairs, or 2.5%. Sequences of E.japonica and E. inquilina differed from each other by 68--72 base pairs, or 11.9-- 12.6 %. E. japonica and E. inquilina were strongly supported (l 00% bootstrap) to be reciprocally monophyletic (Figure 2).

Discussion Our results confirm that E. inquilina and E. japonica are indeed different species according to the biological species concept, which requires that members of a species should be able to reproduce successfully. In our breeding experiments, none ofthe crosses produced healthy planulae, while all of the intra-specific crosses did, corroborating the result of Kubota (1989; 1991b). Additionally, the reciprocal monophyly observed in our phylogeny is consistent with the existence of two different species according to the phylogenetic species concept, which is based on descent (A vise, 2000).

162 Cryptic species have been found in many marine taxa (Knowlton, 1993). Knowlton (1993) distinguishes between sibling species and pseudo-sibling species, where sibling species are morphologically indistinguishable and pseudo-sibling species are distinguishable once the appropriate characters are identified. For Eugymnanthea, the appropriate characters for identification are only present in two features of the medusoid stage (and even at this stage, there are a few exceptions), and the hydro ids are completely indistinguishable. Ecological and biodiversity studies require accurate species-level discrimination (Maurer, 2000), and molecular methods can provide identification when morphology is inadequate (for example, Bucklin et aI., 1995; Hare et aI., 2000). Here, we tested two methods: the mesoglea test (Schmid et aI., 1992) and L6S rDNA sequence comparison. Our results indicate that the mesoglea test does not have sufficient resolution to determine species-level differences, at least for Eugymnanthea. Previously, this test was proven to be effective in species recognition among some, but not all, species of the hydroid Eudendrium tested (Schmid et aI., 1992) Additional tests in other hydroid families are necessary to prove the extent of applicability of this technique. L6S rDNA sequences, however, clearly distinguish E. inquilna and E. japonica. The breeding experiments and DNA sequences also indicate that, unlike the host mussel, the Japanese Eugymnanthea is unlikely to be the result of an introduction from the Mediterranean. The three Japanese specimens, unlike those from the Mediterranean, showed no sequence diversity: all 3 sequences are identicaL. This is probably a result of the small sample size, and is not indicative of a recent invasion, at least not from the

Mediterranean population, whose sequences differ from those of the Japanese about 12%. Our sample sizes for the DNA sequences, while small, are suffcient to reject the hypothesis that the two forms belong to the same population (Hey, 1991). Additionally, the observed 12% divergence falls within the range of interspecific divergences found in other Leptomedusae (Govindarajan, unpublished). Govindarajan et ai. (submitted) calibrated l6S sequence divergence in another Leptomedusae, Obelia geniculata, with the opening of the Bering Strait. Applying their rate of 2.44 x 10-9 substitutions/site/year,

163 which corresponds to 0.49% divergence/milion years, suggests E. inquilna and E. japonica diverged around 24 milion years ago. Assuming the 16S substitution rate in Eugymnanthea is similar (or slower, as that would increase time since divergence), their divergence predates the closure of the Mediterranean from the Pacific in the late Miocene (Por, 1989) which is what would be expected if, as we argue, E. japonica is native to Pacific. Despite our limited sampling, we feel it is highly unlikely that E. japonica is present in the Mediterranean. The hydroid fauna of the Mediterranean is one of the best studied in the world, and it would be surprising if a large population of Eugymnanthea japonica (with its unique morphological and ecological featues) exists there undetected. Indeed, Kubota (1989, in press) examined thousands ofmedusoids, obtained from throughout the Italian coasts. Furthermore, it is probable that the small percentage with the aberrant morphologies is simply intraspecific variation. Considerable intraspecific morphological variability is found in other eirenid medusae. For instance, Kubota (1985;

Kubota, 1991b; Kubota, 1992a; Kubota, 1992b; Kubota, 1993a; Kubota, 1997) discovered through breeding experiments and observations that the 4 forms of eirenid Eutima medusae in Japan belong to the same species (even though one form had features characteristic of a different family!). The absence of Eugymnanthea in other parts of the world, however, may reflect a relict Tethys Sea distribution (Peres, 1985; Boero and Bouillon, 1993) and/or be due to a paucity of studies in those regions. There is a possibility of at least one more species. Salvini-Plawen and Chandrasekhara Rao (1973) described Anthohydra psammobionta, a free-living interstitial hydroid, in the meiofauna from the Bay of Bengal, which, due to the presence of a pedal disk, Salvini- Plawen (l987) later referred to as Eugymnanthea. On the other hand, Bouilon (1985) placed this species in another family, the Olindiasidae

(=Olindiidae) (Limnomedusae). Elucidation of the life cycle and molecular analysis wil be necessary to confirm the placement of this enigmatic species. Kubota (1987a; 2000) discussed the evolutionary origin of Eugymnanthea, speculating that the reduced medusae evolved separately in the Mediterranean and Pacific

164 through independent paedomorphic processes (Boero and Bouilon, 1989). The ancestral form may have had a life cycle like Eutima, with a full medusa stage, and the medusa may have been reduced independently in these two lineages. Recent studies have shown that medusa reduction occurs frequently in Hydrozoa, with the resulting taxonomic implication that this featue should not be used as a generic character, as it curently is in Eugymnanthea and many others (Petersen, 1990; Cunningham and Buss, 1993; Boero et ai. 1997; Govindarajan and Boero, unpublished data (chapter 3D. Thus the presence of the medusoid stage in both E. inquilna and E. japonica does not necessarily imply a close evolutionary relationship. It is likely that either E. japonica was present in Japan before M. galloprovincialis, or that it was introduced from somewhere else in the Pacific. Under the first scenario, E. japonica was present in Japan before M galloprovincialis was introduced, although rare and cryptic. Once introduced (from the Mediterranean or elsewhere), M galloprovincialis provided a better host environment than the native bivalves, allowing E. japonica to increase. The larger overall population of E. japonica may account for the new observations in native bivalve species. Habitat modification may also be causing or contributing to the expansion by providing new habitat for M. galloprovincialis and other host species (Kubota, 1992a). If this scenario is correct, then the increase in E. japonica is an indirect effect of human activities In this scenario, is interesting to speculate about why the hydroids were not introduced along with M galloprovincialis. While it is possible that E. inquilna was introduced to Japan but remains undetected or is represented by the very small percentage of individuals with the Italian morphology (noted above), given the intense study of Japanese bivalve-inhabiting hydroids by Kubota (1983; 1989; 1991a; 1992a; 2003; in press) it seems unlikely that they simply haven't been discovered. Rather, it seems more likely that M galloprovincialis was introduced without the host via introduction of the larval stage in ballast water (Inoue et aI., 1997). As would be expected for a ballast water mediated invasion, the original introduction may have been near the port city of Kobe, and based on a comparison of enzyme polymorphisms with Mediterranean mussels,

165 Wilkens et ai. (1983) concluded that it must have been of a large and continuous natue. Furhermore, based on mitochondrial DNA analysis, Geller et ai. (1994) confirmed the presence of M galloprovincialis larvae in cargo ship ballast water. The second scenario for the origin of E. japonica, that it was introduced from somewhere else in the Pacific, is consistent with the invasive species-like history of E. japonica in Japan. Its relatively recent discovery and subsequent range expansion, and its comparatively lower variation in L6S rDNA sequences and the number of statoliths/statocysts, could be indicative of a recent anthropogenic introduction. However, for the 1 6S rDNA, the lower variation may be a consequence of small sample size. Additional genetic samples and studies in other parts of the Pacific will be necessary to fully evaluate this scenario. This study suggests that DNA sequences will be helpful in identifying crytic species in hydrozoans. DNA sequence analyses will also be useful in identifying invasions (or confirming endemism) in other hydrozoans, as they have been for other marine invertebrates (Wares et ai. 2002). For example, Vainola and Oulasvirta (200l) found used mitochondrial COI sequences to confirm the identity of the introduced limnomedusa, Maeotias marginata, in the Baltic Sea. Other possible hydroid introductions have been speculated; for example Laomedea calceolifera and L. flexuosa in Japan (Chaplygina, 1993), and elytia hummelincki in the Mediterranean (Boero, 2002). These hypotheses could be tested, and the host populations can be identified, using mitochondrial DNA sequences.

Acknowledgements.

We thank F. Boero for use of his laboratory and V. Schmid for direction of the mesoglea test. The Hydrozoan Sequencing Service at Duke University provided some of the DNA sequences for this study. The remainder of the sequencing was conducted in the laboratory of K. Halanych. We thank V Schmid, L. Madin and A. Lindner for comments on the manuscript. G. Fanell offered valuable insights for statistical analysis. A. Govindarajan was supported by WHOI Academic Programs and an NSF PEET grant

166 (DEB--9978131) to C. Cunningham. Financial support to S. Piraino was provided by

MURST(60%, COFIN Projects and FIRB), the Administration of the Province of Lecce, ICRAM (Project "Cnidarian exotic species in the Mediterranean sea"). Support for S. Kubota was provided by the Japan Society for the Promotion of Science and National Research Council ofltaly-CNR. This is contribution No.1 1 180 from the Woods Hole Oceanographic Institution.

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173 Table la. Distrbutions of statoliths per statocyst from a total of 900 1 statocysts observed from medusoids of E. inquilna from Taranto, Italy and 12955 statocysts observed from medusoids of E.japonica (Kubota, 1991a). The distributions are significantly different (Kolmogorov-Smirnov test, p:S 0.01).

# statoliths/statocyst # observations # observations E. inquilna E. japonica 143 149

1 642 1169l 2 4221 958 3 3258 144

4 681 11

5 32 1

6 21 1

7 0 0

8 3 0

174 Table Ib. Morphological overlap in taxonomic characters between E. inquilna and E. japonica. Values for E.japonica are from Kubota (1989; 1991a). Primary diagnostic characters are in bold. For the statoliths per statocyst, the mean, standard deviation, and range (in parentheses) are provided.

Character E. inquilina E. japonica Hydroids Colonial sometimes sometimes Theca absent absent Tentacles 10--30 12--27 Height (mm) 0.2--2.0 0.76--3.5

Medusae Manubrium absent in 97.9%, absent in 2.1 %, present in 2.1 % present in 97.9%

Apical canal sometimes sometimes Gonads 4 4

Radial canals 4 4 Tentacles 0 0 Cirri 0 0 Marginal bulbs 8 8 Statocysts 8 8 Statoliths/statocyst 2.4 :l 0.83 (0--8) 1.08:l 0.36 (0--6) Height (mm) 0.5--1.8 0.59--1. Diameter (mm) 0.7--2.6 0.86--1.

175 Table 2. Results of breeding crosses. The fractions indicate the number of successful matings out of the total number of matings attempted. Healthy planulae are significantly more likely to be produced in intraspecific crosses (Fisher's exact test, p = 0.0018).

E. japonica male E inquilina male

E. japonica female 1/1 0/9 E. inquilina female 0/4 2/2

176 Table 3. Results of the mesoglea tests. The fractions indicate the number of successful adhesions and spreading of cells out of the total number of trials. ECM = Extracellular matrix (mesoglea).

E. inquilina ECM E. japonica ECM P. carnea ECM (Taranto, Italy) (Shirahama, (Naples, Italy) Japan)

E. japonica 1/1 1/1 0/2 (Shirahama, Japan) E. japonica 2/2 Not done 0/2 (Atami, Japan) E. inquilina 2/2 Not done Not done (Taranto, Italy)

177 178 " d b ( C

of -., em , 'I" \ 1/

a c; /G0~ f ,A;'; ¿,~~:' '~_~:.f ~ \ "~ (,. #I

Figure 1. Life cycle of Eugymnanthea. Hydroids (a,b) inhabit bivalves, such as Mytilus galloprovincialis, and asexually produce medusoids (c,d). The medusoids, which are usually separate sexes, are released from the hydroid and produce gametes (e). The gametes fertilize and form planula larvae (f), which settle in a bivalve host (a) and become hydroids. Scale bars: polyp, 250 i-m; medusa, 500 i-m.

179

'\ \ E. japonica 1

B. japonica 2

E. japonica 3

100

B. inquilina 4

B. inquilina 1

99 '- E. inquilÙia 2

- B. inquilina 3 5 changes

Figure. 2. Midpoint-rooted phylogram based on equally-weighted parsimony heuristic search (starting tree through stepwise addition with random addition of taxa (10 replicates), and branch swapping using TBR). Bootstrap numbers are the result of a search with 1000 replicates.

180